Structural insight on the recognition of surface

Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3 Goran Bajica, Laure Yatimea, Robert B. Simb, Thomas Vorup-Jensenc,1, and Gregers R. Andersena,1 Departments of aMolecular Biology and Genetics and cBiomedicine, Aarhus University, DK-8000 Aarhus, Denmark; and bDepartment of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom Edited by Douglas T. Fearon, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, and approved August 28, 2013 (received for review June 13, 2013)

Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin αMβ2, or CD11b/CD18) is expressed on all macrophages and recognizes iC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of iC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for iC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site–bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d: CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes. innate immunity

| phagocytosis | integrin receptor | structural biology

A

ctivation of complement leads to proteolytic cleavage of the central complement component, C3. Its major fragment, C3b, acts as an opsonin and becomes covalently bound to the activating surface via a reactive thioester located in the thioester (TE) domain of nascent C3b (Fig. S1A). Proteolytic processing by factor I within the CUB domain of C3b leads to the formation of iC3b and C3dg. Finally, C3d—which practically corresponds to the TE domain present in C3, C3b, and iC3b (Fig. S1 B–G)— is formed by other plasma proteases. These activation products are ligands for five complement receptors (1), with iC3b being the primary ligand of complement receptors (CRs) CR3 and CR4 (also known as CD11c/CD18, p150,95, or integrin αXβ2), which is structurally similar to CR3. Like other integrins, CR3 is a heterodimeric complex of two transmembrane proteins, αM and β2. It is abundantly expressed on myeloid leukocytes, including neutrophil granulocytes, dendritic cells, monocytes, and macrophages and also on lymphoid natural killer (NK) cells (2). Most ligands, including iC3b (3), are bound by the Von Willebrand factor A (VWA) domain in the α-chain, also referred to as the αI domain owing to its insertion in the β-propeller domain. I domain residues coordinate a metal ion essential for ligand recognition through a metal ion-dependent adhesion site (MIDAS). Integrins adopt at least three major conformations in the cell membrane. The bent-closed conformation is inactive in ligand binding, the extended-closed conformation has low ligand affinity, and the extended-open conformation binds ligands with high affinity. The transition from the bentclosed to the open-extended conformation is exerted by a cytoplasmic force on the leg of the β-subunit, a process usually referred to as the inside-out signaling (4). Binding of ligands to CR3 leads to conformational changes in its ectodomain transmitting an outside-in signal through the cell membrane. This may lead to actin remodeling, phagocytosis, 16426–16431 | PNAS | October 8, 2013 | vol. 110 | no. 41

degranulation, and changes in leukocyte cytokine production (2, 5–7). CR3, and to a lesser degree CR4, are essential for the phagocytosis of complement-opsonized particles or complexes (6, 8, 9). Complement-opsonized immune complexes are captured in the lymph nodes by CR3-positive subcapsular sinus macrophages (SSMs) and conveyed directly to naïve B cells or through follicular dendritic cells (10) using CR1, CR2, and Fcγ receptors for antigen capture (11, 12). Hence, antigen-presenting cells such as SSMs may act as antigen storage and provide B lymphocytes with antigens (10, 12). Here, we establish the C3d fragment as the minimal and highaffinity binding partner for the CR3 I domain. By contrast, the binding site for the CR4 I domain was located in the C3c fragment by electron microscopy (13). We present the crystal structure of the CR3 I domain in complex with C3d. The classic observation of CR3 binding to iC3b, but not to its precursor C3b (14), is consistent with our structure. In addition, our structure and functional data suggest simultaneous binding of CR3 and another complement receptor, CR2, to C3 fragments, which might provide the basis for trafficking of complement-opsonized immune complexes from macrophages to B cells and follicular dendritic cells in lymph nodes. Results CR3 and CR4 I Domains Recognize Distinct Binding Sites on iC3b. To

quantitatively compare binding properties of the CR3 and CR4 I domain with regard to binding of C3 proteolytic fragments, C3b, Significance Fragments of complement component C3 tag surfaces such as those presented by microbial pathogens or dying host cells for recognition by cells from the innate immune system. Complement receptor (CR) 3 enables efficient binding of complementtagged surfaces by macrophages and dendritic cells, which eventually transport the CR3-bound material into lymph nodes. The study identifies in atomic details the fragments of CR3 and C3 required for such binding. The structural organization permits concomitant recognition by another complement receptor, namely CR2, expressed on cells of the adaptive immune system, suggesting a structural rationale for the exchange of antigens between leukocytes of the innate and adaptive immune systems critical in the formation of humoral immune responses. Author contributions: G.B., L.Y., T.V.-J., and G.R.A. designed research; G.B. and T.V.-J. performed research; R.B.S. contributed new reagents/analytic tools; G.B., L.Y., T.V.-J., and G.R.A. analyzed data; and T.V.-J. and G.R.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: Crystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4M76). 1

To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1311261110/-/DCSupplemental.

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Crystal Structure of CR3 I Domain in Complex with C3d. Guided by the quantitative investigations made above, we determined the crystal structure of the CR3 I domain (subunit αM residues 127– 321, mature numbering) in complex with C3d (C3 residues 993– 1,288, prepro numbering) at 2.8 Å resolution (Table S1 and Fig. S4A). The structure reveals a 1:1 complex between the CR3 I domain and C3d (Fig. 2A). The integrin I domain folds into an α/β Rossmann fold and adopts its open conformation as shown by comparison with the open-conformation structure of the I domain (Fig. S4B). The I domain α7 helix is shifted toward its C terminus, and this conformation is most likely favored by the I316G mutation introduced for this purpose. The open conformation is likewise adopted in the MIDAS site, where the hexacoordinated metal ion is coordinated by Ser142, Ser144, Thr209 and two water molecules (Fig. 2B), whereas the last coordination position is occupied by an aspartate from C3d. Because Mg2+ was not compatible with the electron density in the MIDAS site, and because Ni2+ was present in the crystallization buffer, we used anomalous diffraction data to confirm the presence of a Ni2+

log 10 kd CR4

I domain

CR3

0

C3b

A

iC3b

B

ion in the MIDAS site (Fig. 2B and Table S1). The ability of Ni2+ to stabilize MIDAS interactions with a ligand is well known from the complement convertases (18). Within the complex, C3d adopts the well-described compact α-α6 barrel structure (Fig. 2A and Fig. S4C) with only minor conformational differences compared with known structures containing C3d (19–21). The α-helix connecting loop regions of C3d are presented in an alternating fashion at the circumference of two opposite surfaces: a concave, mainly negatively charged surface and a positively charged convex surface. At the rim of the concave surface Asp1247, situated in a loop region connecting helices α10 and α11 (Fig. 2C and Fig. S4 C and D), provides the final coordination bond to the divalent cation in the CR3 MIDAS (Fig. 2B). The quite polar interface between C3d and the I domain is modest, with an area of ∼490 Å2, which is smaller but comparable to similar I domain:ligand complexes (Table S2). Besides Asp1247 on C3d involved in the MIDAS ion interaction, the nearby Asp1245 in C3d engages in a salt bridge with CR3 Arg208 (Fig. 2C and Fig. S4D). C3d Lys1217 also seems to stabilize the interaction, because it is capable of forming salt bridges with CR3 Glu178 and Glu179 and hydrogen bonds with main chain carbonyls of Leu205 and Leu206. Finally, C3d Arg1254 also contributes to the interaction by forming hydrogen bonds with main-chain carbonyls of Gly143 and Ile145 in the I domain via a water molecule. Conserved Features of MIDAS-Dependent Ligand Interactions. Besides iC3b, the CR3 I domain is responsible for interacting with a variety of ligands, including fibrinogen, ICAM-1, and RAGE (22), but structures of their I domain complexes are not available. To identify general features among ligand side chains engaging in MIDAS ion coordination that might promote the identification of I domain interacting residues in other CR3 ligands, we identified all unique structures containing a VWA domain or a βI domain engaging in a MIDAS-dependent interaction with a ligand protein. Comparison of these structures revealed obvious trends for the use of either aspartate or the longer glutamate side chains as MIDAS ligands. If the small aspartate side chain is used, as in the C3d complex, it is located in a loop region or at the termini of a peptide (Fig. S5 A–D). This is even true for complexes between antibody Fab fragments and I domains (Fig. S5 E–G), where an aspartate is located peripherally in the heavy chain CDR3 loop. Even smaller than the aspartate and located at the end of a flexible region is the exposed C-terminal carboxylate group in the noncatalytic subunit of complement convertases (C3b/cobra venom factor/C4b) coordinating the MIDAS ion in the VWA domain (Fig. S5H) of the catalytic subunit (factor B/C2). We found three unique examples involving the longer glutamate side chain (Fig. S5 I–K). In complexes between the LFA-1 I domain and ICAM-1/3/5, a glutamate

C3d

C

−1

−2

−3 0

E

D

F

−1

−2

−3

−8 −7 −6 −5 −4 −3 −2 −1 −8 −7 −6 −5 −4 −3 −2 −1 −8 −7 −6 −5 −4 −3 −2 −1

log10 KD Bajic et al.

RU 1000 900 800 700 600 500 400 300 200 100 0 500 450 400 350 300 250 200 150 100 50 0

Fig. 1. SPR analysis of the C3 fragment binding selectivity of the CR3 (A–C) and CR4 I (D–F) domains. The CR3 or CR4 I domain, stabilized by mutagenesis in the open, ligand-binding conformation (50, 51), were injected in concentrations ranging from 250 nM to 100 μM over surfaces coupled with C3b (A and D), iC3b (B and E), or C3d (C and F). The data were analyzed with the EVILFIT algorithm with settings assuming a priori all binding parameters to be equally likely (52, 53). The volume of interactions, indicated with colored contours (in RU as shown by scale bars) was plotted as a function of the dissociation constant (10−8 M ≤ KD ≤ 10−1 M) and rate (10−3 s−1 ≤ kd ≤ 100 s−1). Red arrows indicate a population of high-affinity interactions for the CR3 I domain (KD ∼0.4 μM) shared between iC3b and C3d but not observed for interactions with C3b.

PNAS | October 8, 2013 | vol. 110 | no. 41 | 16427

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iC3b, C3c, C3dg, and C3d were immobilized in surface plasmon resonance (SPR) flow cells. For both I domains good binding signals were observed with C3b, iC3b, and C3c as ligands (Fig. S2 A–C and F–H). Nevertheless, even high concentrations (100 μM) of the CR3 or CR4 I domain did not lead to saturation. This is consistent with X-ray crystallography (15) and inhibition experiments showing that both the CR3 and CR4 I domain interact weakly (Kd ∼300 μM) with acidic side chains acting as ligand mimetics (16). The C3d and C3dg-coated surfaces produced robust SPR signals of ∼1,200 resonance units (RU) and showed signs of saturation at high CR3 I domain concentrations (Fig. S2 D and E). By contrast, the CR4 I domain only poorly bound these fragments (Fig. S2 I and J). As detailed in other studies (16, 17), the binding kinetics of the CR3 and CR4 I domain ligand binding are not well-described with simple 1:1 Langmuir isotherms. The interactions were quantified by analysis of the sensorgrams with the EVILFIT algorithm, which calculates the minimal distribution in binding kinetics for the heterogeneous interactions with ligands (Fig. 1). In general, the modeled distribution in kinetics efficiently described the experimental data as reflected in the small rmsds. For the CR3 and CR4 I domain, some of the interactions with C3b, iC3b, and C3c were of modest strength with Kd ∼10−4 to 10−3 M (Fig. 1 A and B and D–F), that is, quantitatively equivalent to binding of acidic side chains reported earlier (16). However, the classic CR3 ligand iC3b presented a population of interactions with Kd ∼10−7 to 10−6 M (Fig. 1B), not found with either C3b or C3c or for any fragments probed with the CR4 I domain (Fig. S3). This high-affinity type of interaction dominated the binding of the CR3 I domain to C3dg and C3d (Fig. 1 and Fig. S3).

A

α7

C-terminal

B

CR3 I-domain α5

Ni2+ C-terminal

N-terminal α1

Asp 242 Ser 142 Thr 209

α5 N-terminal

Ser 144

C3d

Asp 1247

α3

Q1013

C

Glu179 Leu205

45°

Ile145

Glu178

Glu179

Leu205 Ile145

Glu178 Arg208

Asp1247

Gly143 Lys1217

Arg1254

Lys1217

Leu206 Gly143

Asp1245

side chain at the end of a β-strand in an ICAM Ig domain is a MIDAS ion ligand. A resembling situation is found for the α2β1 I domain:collagen complex. In both cases the glutamate is protruding from a region containing regular secondary structure with little curvature and flexibility that would permit a closer approach to the MIDAS ion and the use of the shorter aspartate. In a third case, a crystal-packing interaction by a glutamate located immediately after an α-helix mimics a ligand of the CR3 I domain (15). Overall, there seems to be a steric selectivity in the MIDAS ion coordination by ligand acidic groups: An aspartate side chain is preferred in loops or flexible termini and the longer glutamate is preferred in regions with secondary structure, whereas both aspartate and glutamate located next to secondary structure are possible MIDAS ion coordinators. C3d Asp1247 Is Essential for CR3 I Domain Interaction. Because integrins may promiscuously bind acidic residues exposed on protein surfaces (15, 16, 22) and to exclude crystal-packing artifacts, we mutated C3d residues in the intermolecular interface. We tested iC3b and WT or mutated C3d for their ability to interact with CR3 I domain by isothermal titration calorimetry (ITC) experiments (Fig. 3). All of the binding ligands interacted with the CR3 I domain in a 1:1 stoichiometry, and iC3b generated from C3b with factor I bound the I domain with a KD of 600 nM. The I domain bound WT C3d with a KD of 450 nM. These numbers are in good agreement with the high-affinity site identified by SPR (Fig. 1 B and C and Fig. S3B). Mutation of C3d Asp1247 to alanine abolished CR3 I domain binding to C3d, suggesting a crucial role of this aspartate in the interaction with the MIDAS-coordinated cation. Replacing Asp1247 by a glutamate also impaired the interaction, showing that simple conservation of charge is not sufficient (Fig. 3). Most likely, when the longer glutamate side chain in this C3d mutant interacts with the I domain MIDAS site the adjoining interactions in the interface are difficult to form. The R1254A mutant (Fig. 3) displayed a lowered affinity of 2.2 μM, whereas the mutant C3d proteins K1217A and K1217A/ R1254A showed no detectable binding. Together, these data indicate that C3d Asp1247 is essential for the interaction with CR3 I domain but is not sufficient, because other residues of the opposite charge, namely K1217 and R1254, are required to steer the interaction. Physiological Significance of the C3d:I Domain Structure. The in vitro confirmation of our crystal structure by ITC and SPR experiments is also supported by prior data, because the iC3b binding site on the CR3 I domain earlier suggested (23) is in excellent 16428 | www.pnas.org/cgi/doi/10.1073/pnas.1311261110

Arg1254

Fig. 2. The structure of the C3d:I domain complex. (A) The edge of C3d (brown) interacts with the MIDAS (marked by the Ni2+ ion) of the CR3 I domain (purple). (B) The Ni2+ ion bound in the MIDAS. The electron density contoured at 6 σ obtained from anomalous differences in diffraction data (Table S1) is shown as a mesh around the Ni2+ ion. (C) Details of the intermolecular interface with putative hydrogen bonds and electrostatic interactions indicated by dashed lines. C3 labels are underlined in all panels. Fig. S4D shows a stereo view of the interface.

agreement with our structure. Furthermore, the participating residues are strictly or highly conserved in mammalian C3 and CR3 αM sequences (Fig. S6A). In contrast, none of the C3 residues involved in the I domain interaction is conserved in the structural C3 homolog C4 (Fig. S6B) (24), also being cleaved to C4b and undergoing further degradation to iC4b and C4d. Likewise, αM residues with side chains interacting with C3d (Arg208, Glu178, and Glu179) are not conserved in CR4. Importantly, our structure also offers an explanation for why the iC3b proteolytic stage must be reached to allow CR3 interaction (25). The CUB domain in C3b connects the TE domain to the C3c moiety of C3b, and proteolytic degradation of the CUB domain probably causes its partial collapse in the resulting iC3b (Fig. S1E). Superposition of known structures of C3b suggests that the CUB domain prevents the I domain from interacting with the TE domain by steric hindrance (Fig. 4). Finally, our structure is in accordance with a favorable geometry where the CR3 binding site of iC3b or C3d(g) opsonizing the activator is accessible for a phagocytotic CR3-presenting cell. The CR3 binding site is separated by 40 Å from the Gln1013 forming the covalent bond to the complement activator surface (Fig. 4A). In conclusion, the in vivo relevance of our structure is strongly supported by (i) SPR and ITC experiments quantitating the interaction of iC3b, WT C3d, and mutated C3d with the I domain, (ii) the conservation of the residues in the molecular interface, (iii) our ability to rationalize the discrimination against CR3 binding to C3b, and (iv) the inferred lack of steric hindrance imposed by the activator upon CR3 binding. C3 Thioester Domain As a Molecular Hub. Our structure of the C3d:I domain complex together with the structures of C3 (26, 27), the C3d complexes with factor H (20, 28), and CR2 (21) demonstrate that a substantial fraction of the surface of the C3 TE domain is involved in intra- or intermolecular contacts in one of the multiple functional states of C3. Strikingly, surface residues on the TE domain interacting with other domains in native C3 are almost perfectly separated from those surface patches forming contacts with fH, CR3, and CR2 in their C3d complexes. In C3, the convex surface of C3d forms contacts with the MG2, CUB, and MG8 domains (total interface area of ∼2,650 Å2) that bury more than 20% of the TE domain surface (Fig. 5A). On the circumference and the rim of the concave surface of C3d, surface patches interacting with fH (∼600 Å2), CR3 (∼500 Å2), and CR2 (∼1,100 Å2) are likewise almost perfectly nonoverlapping with each other and with the above-mentioned C3 interdomain interface (Fig. 5 B and C). Hence, especially in the iC3b state, the Bajic et al.

80 90

K D = 0.45 μM

K D = 0.6 μM

molar ratio iC3b:I domain

molar ratio C3d WT:I domain

K D = 2.2 μM

molar ratio C3d R1254A:I domain

molar ratio C3d D1247E:I domain

Fig. 3. ITC studies of the CR3 I domain interaction with iC3b, C3d WT, C3d R1254A, and C3d D1247E mutants. Raw titration isotherms and the integrated peak areas are shown. Dissociation constants calculated according to a simple independent binding site model are displayed.

Simultaneous Binding of CR2 and CR3 to iC3b. CR2 binds with similar affinity to iC3b and C3d(g) and with C3d as the minimal ligand (29). To verify whether the formation of the CR2:C3d: CR3 complex suggested above is possible, C3d and CR2 complement control protein (CCP) domains 1-2 were subjected to pull-down experiments with immobilized CR3 I domain. Bound proteins were eluted with EDTA to disrupt MIDAS–ligand interactions. In the presence of WT C3d, both CR2 and C3d were eluted from the CR3 affinity column, demonstrating that the CR3 I domain and CR2 CCP 1-2 are able to simultaneously bind to C3d (Fig. 5D and Fig. S7). Using instead the C3d D1247A mutant no protein was eluted, showing that CR2 is not interacting nonspecifically with the resin and that this C3d aspartate is essential for interaction with the CR3 MIDAS. With the C3d mutant D1154A unable to bind CR2 (30), CR2 was virtually absent from the EDTA eluate (Fig. 5D and Fig. S7). To

C345C

A

B CUB

Nt-α’

in

ma

o βI-d

I-domain β-propeller -propellerr

Ni2+ TE

C3b

Q1013

~40 Å

Activator surface Fig. 4. The CR3 I domain discriminates against C3b. (A) The structure of C3b superimposed with the C3d:CR3 I domain complex. The βI domain and the α-chain β-propeller within CR3 are shown schematically. (B) Close-up of the region framed in A. Notice the overlap between the CR3 I domain and the C3b CUB domain in the hypothetical CR3:C3b complex. C3 labels are underlined.

Bajic et al.

further corroborate the existence of the ternary CR2:C3d:CR3 complex, size-exclusion chromatography experiments were conducted (Fig. S8). The chromatograms and SDS/PAGE analysis of fractions from these experiments showed that the complexes elute in the expected order CR3:C3d:CR2, CR3:C3d, and CR2: C3d (Fig. 5E and Fig. S8). Importantly, the presence of CR2 in the early fractions from the CR3:C3d:CR2 experiment can only be explained by the fact that CR2 was engaged in the ternary complex together with C3d and CR3. In conclusion, our pulldown and size-exclusion chromatography experiments confirmed the existence of a ternary complex in which the CR3 I domain and the CR2 CCP 1-2 fragment bind simultaneously to C3d. Discussion The engulfment by immune cells of complement-opsonized objects is a fundamental property of the human immune defense where CR3 plays an essential role (8). CR3’s specificity for iC3b as ligand has been established for 30 years. Now, our results define where and how CR3 interacts with iC3b and further emphasize the astonishing central role of the C3d moiety in intermolecular contacts. The CR3 I domain is well established as the primary binding site for iC3b (3, 31). The quantitative findings in our study clearly suggest that the binding of the CR3 I domain to C3d is 100- to 500-fold stronger than its weaker binding of simple acidic groups. Hence, the structure of the C3d: CR3 I domain complex shows a type of interaction distinct from the earlier report showing the binding to a ligand mimetic, that is, glutamate side chain contributed by a crystal lattice contact (15). Nevertheless, although the I domain is an important determinant in the binding of iC3b and C3d(g), other regions in CR3 must contribute, because deletion of its I domain results in residual iC3b affinity (32). Both the αM β-propeller and the β2 Ilike domain (Fig. 4A) have been suggested to be implicated in the interaction with iC3b (33–35). On the iC3b side, mutations in the Nt-α′ region weaken the iC3b–CR3 interaction (36). The degradation product C3d(g) is not normally considered as a CR3 ligand, and soluble C3d cannot outcompete erythrocyte-bound iC3b in a rosette assay (37, 38). However, coating of erythrocytes with C3d facilitates their phagocytosis by monocytes in a metal ion- and CR3-dependent manner, although C3d does this much less efficiently than iC3b (39). This is likely to directly mirror the interaction we observe in the crystal structure and have quantitated by SPR and ITC. In summary, there seem to be at least two contact points between iC3b and CR3, a crucial one involving the C3d:I domain interaction established by us. If the iC3b Nt-α′ region in the C3c moiety of iC3b and the αM β-propeller and the β2 I-like domain in CR3 indeed are important for the receptor– PNAS | October 8, 2013 | vol. 110 | no. 41 | 16429

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TE domain may bind simultaneously and strongly to the two complement receptors and collateral binding of factor H and CR3 seems possible as well.

A

C Q1013

C3 180

CR3 I-domain

Ni2+

o

CR2 CCP 1-2 C3d

B

CR3

CR2 fH

fH CCP 19-20

D Input Mw C3d WT proteins kDa C3d CR2 50 FT W FT W Elute

C3d D1247A

C3d D1154A

FT W FT W Elute FT W FT W Elute

40 30 25 20 15 10

E

Mw

kDa 50 40 C3d 30 25 CR3 CR2 20 15

C3d:CR2

32 33 34 35

C3d:CR3

CR3:C3d:CR2

32 33 34 35 32 33 34 35 Fraction number

Fig. 5. The multiple interactions of the TE domain in C3 and its proteolytic degradation products. (A) Surface areas (gray) in the C3 TE domain interacting with the CUB, MG8, and the MG2 domains in native C3 are mapped onto C3d from its CR3 complex; the thioester Gln1013 is shown in red. (B) As in A after a 180° rotation. Surface areas of C3d interacting with CR2 (pink), factor H (green), and CR3 (purple) are indicated. (C) Superposition of the complex between C3d (brown surface) with the CR3 I domain (purple cartoon) with that of the C3d:CR2 complex (21) and the C3d:factor H complex (20). (D) Silver-stained gel after denaturing SDS/PAGE analysis of fractions from the CR3 I domain affinity column. Labels + and − indicate, respectively, the presence or absence of the protein in the pull-down assay at that particular step. From left to right, molecular weight (Mw) marker together with purified CR2 CCP 1-2 (insect cell-expressed) and C3d and CR3 I domain affinity column pull-down using C3d WT showing the flow-through and the wash steps and the final EDTA elution. The same fractions are shown for the C3d D1247A and the D1154A control mutants unable to bind CR3 and CR2, respectively. All the volumes in wash and elution steps were the same. Likewise, the volumes loaded for SDS/PAGE analysis were identical. For comparison, the pull-down was performed with the CR2 expressed in either bacteria or insect cells (Fig. S7). (E) Silver-stained gel after denaturing SDS/ PAGE analysis of fractions from analytical size-exclusion chromatography (Fig. S8). The same fractions resulting from the C3d:CR2, C3d:CR3 and CR3: C3d:CR2 runs were analyzed.

16430 | www.pnas.org/cgi/doi/10.1073/pnas.1311261110

ligand interactions, contacts involving these are likely to be spatially well separated from the C3d:I domain interface (Fig. 4A). Our model of iC3b binding to CR3 is clearly distinct from that proposed for CR4 by prior EM studies (13). In side-by-side SPR experiments of the I domain binding of C3 fragments, the iC3b and C3d(g) fragments presented high-affinity binding sites for the CR3 I domain, whereas C3b and C3c had no such interactions. These findings corroborate earlier studies on the intact receptor, supporting the relevance of studying the I domain selectivity towards C3 fragments. The CR4 I domain bound with almost indiscriminate kinetics to C3b, iC3b, and C3c, whereas the C3d(g) fragments were much poorer ligands. This is also in close agreement with the work by Chen et al. (13) on the intact ectodomain of CR4, which identified a major binding site for this receptor involving the MG3 and MG4 domains of C3c. The orientation of these domains, and hence the binding interface for CR4, are conserved between C3b and C3c (40), likely also making this the case for iC3b. This is quantitatively supported by the nearly identical kinetics for the CR4 I domain binding of these fragments observed in our study. Our finding that CR2 and the CR3 I domain can bind to the same molecule of C3d is intriguing, and because CR2 does not seem to recognize elements outside the C3d moiety of iC3b, our results imply that one iC3b or C3d(g) molecule on soluble immune complexes can bridge a CR3-presenting cell with a CR2presenting cell. The location of the ligand-binding region (CR2 CCP 1-2 and CR3 I domain) at the distant end of these large receptors far from the cell membrane makes immune complex bridging between two cells a realistic scenario. One important example is the suggested hand-over of complement-opsonized immune complexes from CR3-bearing subcapsular sinus macrophages to CR2 on naïve B cells or follicular dendritic cells in lymph nodes (10, 12). Concerning the remaining complement receptors, the binding site for CR4 at the MG3–MG4 domains mapped by EM (13) seems not to overlap with regions implicated in binding CR3. Both receptors are expressed together on many cell types including macrophages, monocytes, neutrophils, and NK cells (2), suggesting that simultaneous CR3 and CR4 binding to the same iC3b molecule is possible. The CRIg receptor expressed on a subset of tissue macrophages recognizes C3b, iC3b, and C3c, and its binding site has been mapped to the MG domains 3, 4, 5, and 6 and the LNK regions of C3b (41). Because the structure of iC3b is only known to low resolution and is controversial (13), the exact spatial relation between the iC3b binding sites for CRIg and the CR3 I domain found by us is unknown but likely to be variable. However, these sites are nonoverlapping, suggesting that both receptors could bind simultaneously to iC3b unless other parts of CR3 compete with CRIg. With respect to complement receptor 1, both CR1 and CR3 binding involve the same part of the Nt-α′ region (36), suggesting mutually exclusive binding of the two receptors to the same iC3b molecule. Therapeutic intervention through blockade of CR3 has been suggested for treatment of cerebral stroke (42). Furthermore, macrophage migration to the brain is likely to involve CR3 (43). However, owing to the diversity of CR3 ligands, their central role in immune clearance of pathogens and apoptotic cells by phagocytosis (8, 44), adhesion (45, 46), and transmigration (47) overall or even function-specific CR3 blockade is not without risks. As an example, systemic lupus erythematosus is tightly linked with a single mutation in the αM chain of CR3 that mainly affects its role in phagocytosis (48). In the same manner, C3b and its proteolytic fragments also engage in interactions with a large number of other proteins, thereby severely complicating the development of complement inhibitors. Our structure of the C3d:I domain complex provides an important contribution to the development of more selective complement inhibitors by identifying the surface areas on the CR3 I domain and on the C3d moiety of importance for their mutual interaction. Owing to the proximity of the C3d–CR2 interaction it may also contribute to improve the CR2-based targeting strategies under development for site-specific delivery of complement inhibitors (49). Bajic et al.

WT or mutated C3d and CR3 I domain were prepared as recombinant proteins in Escherichia coli. The CR2 CCP 1-2 fragment was prepared as recombinant protein in insect cells or E. coli. C3b was prepared by trypsin digestion of C3, and iC3b, C3d(g), and C3c were prepared from C3b by factor I digestion in the presence of fH. In the SPR experiments proteolytic C3 derivatives were coupled through primary amine groups to the CM4 sensor chip and the CR3 I or CR4 I domain injected over the chip. ITC measurements were performed at 25 °C by titration of CR3 I domain with iC3b or WT/mutated C3d. For the C3d pulldown experiments, the CR3 I domain was coupled to CNBr-activated Sepharose, and C3d and CR2 were bound in the presence of Mg2+ and eluted with EDTA. The C3d:I domain complex was crystallized by vapor diffusion. Diffraction data from frozen crystals were collected with synchrotron radiation at the Swiss Light Source or European Synchrotron Radiation Facility, and the structure of

1. van Lookeren Campagne M, Wiesmann C, Brown EJ (2007) Macrophage complement receptors and pathogen clearance. Cell Microbiol 9(9):2095–2102. 2. Ross GD (2000) Regulation of the adhesion versus cytotoxic functions of the Mac-1/ CR3/alphaMbeta2-integrin glycoprotein. Crit Rev Immunol 20(3):197–222. 3. Diamond MS, Garcia-Aguilar J, Bickford JK, Corbi AL, Springer TA (1993) The I domain is a major recognition site on the leukocyte integrin Mac-1 (CD11b/CD18) for four distinct adhesion ligands. J Cell Biol 120(4):1031–1043. 4. Springer TA, Dustin ML (2012) Integrin inside-out signaling and the immunological synapse. Curr Opin Cell Biol 24(1):107–115. 5. Lefort CT, et al. (2009) Outside-in signal transmission by conformational changes in integrin Mac-1. J Immunol 183(10):6460–6468. 6. Dupuy AG, Caron E (2008) Integrin-dependent phagocytosis: Spreading from microadhesion to new concepts. J Cell Sci 121(Pt 11):1773–1783. 7. Shimaoka M, Takagi J, Springer TA (2002) Conformational regulation of integrin structure and function. Annu Rev Biophys Biomol Struct 31:485–516. 8. Underhill DM, Ozinsky A (2002) Phagocytosis of microbes: Complexity in action. Annu Rev Immunol 20:825–852. 9. Wright SD, Meyer BC (1986) Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes. J Immunol 136(5):1759–1764. 10. Phan TG, Grigorova I, Okada T, Cyster JG (2007) Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat Immunol 8(9):992–1000. 11. Gray EE, Cyster JG (2012) Lymph node macrophages. J Innate Immun 4(5–6):424–436. 12. Gonzalez SF, et al. (2011) Trafficking of B cell antigen in lymph nodes. Annu Rev Immunol 29:215–233. 13. Chen X, Yu Y, Mi LZ, Walz T, Springer TA (2012) Molecular basis for complement recognition by integrin αXβ2. Proc Natl Acad Sci USA 109(12):4586–4591. 14. Beller DI, Springer TA, Schreiber RD (1982) Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor. J Exp Med 156(4):1000–1009. 15. Lee JO, Rieu P, Arnaout MA, Liddington R (1995) Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell 80(4):631–638. 16. Vorup-Jensen T, et al. (2005) Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin alphaXbeta2. Proc Natl Acad Sci USA 102(5):1614–1619. 17. Vorup-Jensen T (2012) Surface plasmon resonance biosensing in studies of the binding between β₂ integrin I domains and their ligands. Methods Mol Biol 757:55–71. 18. Fishelson Z, Müller-Eberhard HJ (1982) C3 convertase of human complement: Enhanced formation and stability of the enzyme generated with nickel instead of magnesium. J Immunol 129(6):2603–2607. 19. Nagar B, Jones RG, Diefenbach RJ, Isenman DE, Rini JM (1998) X-ray crystal structure of C3d: A C3 fragment and ligand for complement receptor 2. Science 280(5367):1277–1281. 20. Morgan HP, et al. (2011) Structural basis for engagement by complement factor H of C3b on a self surface. Nat Struct Mol Biol 18(4):463–470. 21. van den Elsen JM, Isenman DE (2011) A crystal structure of the complex between human complement receptor 2 and its ligand C3d. Science 332(6029):608–611. 22. Vorup-Jensen T (2012) On the roles of polyvalent binding in immune recognition: Perspectives in the nanoscience of immunology and the immune response to nanomedicines. Adv Drug Deliv Rev 64(15):1759–1781. 23. Ustinov VA, Plow EF (2005) Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2. Biochemistry 44(11):4357–4364. 24. Kidmose RT, et al. (2012) Structural basis for activation of the complement system by component C4 cleavage. Proc Natl Acad Sci USA 109(38):15425–15430. 25. Ross GD, Medof ME (1985) Membrane complement receptors specific for bound fragments of C3. Adv Immunol 37:217–267. 26. Fredslund F, et al. (2006) The structure of bovine complement component 3 reveals the basis for thioester function. J Mol Biol 361(1):115–127. 27. Janssen BJ, et al. (2005) Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437(7058):505–511. 28. Kajander T, et al. (2011) Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost discrimination by complement. Proc Natl Acad Sci USA 108(7): 2897–2902. 29. Diefenbach RJ, Isenman DE (1995) Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement

Bajic et al.

the complex was determined by molecular replacement using Protein Data Bank ID codes 1IDO and 1C3D as search models. Coordinates and structure factors have been deposited at the Protein Data Bank with ID code 4M76. Detailed methods and the associated references can be found in SI Methods. ACKNOWLEDGMENTS. We thank T. Springer for inspiration and advice, Y. He and the beamline staff at the Swiss Light Source and European Synchrotron Radiation Facility for help with data collection, A.M. Bundsgaard and B. W. Grumsen for technical assistance, and J. K. Jensen and L. T. Pallesen for help with isothermal titration calorimetry experiments. This work was supported by the Lundbeck Foundation through the Lundbeck Foundation Nanomedicine Center, the MEMBRANES center, and the Novo-Nordisk Foundation through a Hallas-Møller Fellowship (to G.R.A.). T.V.-J. was supported by the Carlsberg Foundation, the LEO Foundation, Helga og Peter Kornings Fond, and Gluds Legat.

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35. 36.

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receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2. J Immunol 154(5):2303–2320. Clemenza L, Isenman DE (2000) Structure-guided identification of C3d residues essential for its binding to complement receptor 2 (CD21). J Immunol 165(7):3839–3848. Ueda T, Rieu P, Brayer J, Arnaout MA (1994) Identification of the complement iC3b binding site in the beta 2 integrin CR3 (CD11b/CD18). Proc Natl Acad Sci USA 91(22): 10680–10684. Yalamanchili P, Lu C, Oxvig C, Springer TA (2000) Folding and function of I domaindeleted Mac-1 and lymphocyte function-associated antigen-1. J Biol Chem 275(29): 21877–21882. Li Y, Zhang L (2003) The fourth blade within the beta-propeller is involved specifically in C3bi recognition by integrin alpha M beta 2. J Biol Chem 278(36):34395–34402. MacPherson M, Lek HS, Prescott A, Fagerholm SC (2011) A systemic lupus erythematosusassociated R77H substitution in the CD11b chain of the Mac-1 integrin compromises leukocyte adhesion and phagocytosis. J Biol Chem 286(19):17303–17310. Xiong Y-M, Haas TA, Zhang L (2002) Identification of functional segments within the beta2I-domain of integrin alphaMbeta2. J Biol Chem 277(48):46639–46644. Taniguchi-Sidle A, Isenman DE (1994) Interactions of human complement component C3 with factor B and with complement receptors type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) involve an acidic sequence at the N-terminus of C3 alpha’-chain. J Immunol 153(11):5285–5302. Schreiber RD, Pangburn MK, Bjornson AB, Brothers MA, Müller-Eberhard HJ (1982) The role of C3 fragments in endocytosis and extracellular cytotoxic reactions by polymorphonuclear leukocytes. Clin Immunol Immunopathol 23(2):335–357. Ross GD, Lambris JD (1982) Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes. J Exp Med 155(1):96–110. Gaither TA, Vargas I, Inada S, Frank MM (1987) The complement fragment C3d facilitates phagocytosis by monocytes. Immunology 62(3):405–411. Janssen BJ, Christodoulidou A, McCarthy A, Lambris JD, Gros P (2006) Structure of C3b reveals conformational changes that underlie complement activity. Nature 444(7116): 213–216. Wiesmann C, et al. (2006) Structure of C3b in complex with CRIg gives insights into regulation of complement activation. Nature 444(7116):217–220. Zhang L, et al. (2003) Effects of a selective CD11b/CD18 antagonist and recombinant human tissue plasminogen activator treatment alone and in combination in a rat embolic model of stroke. Stroke 34(7):1790–1795. Riou A, et al. (2013) MRI assessment of the intra-carotid route for macrophage delivery after transient cerebral ischemia. NMR Biomed 26(2):115–123. Morelli AE, et al. (2003) Internalization of circulating apoptotic cells by splenic marginal zone dendritic cells: dependence on complement receptors and effect on cytokine production. Blood 101(2):611–620. Diamond MS, et al. (1990) ICAM-1 (CD54): A counter-receptor for Mac-1 (CD11b/ CD18). J Cell Biol 111(6 Pt 2):3129–3139. Chavakis T, et al. (2003) The pattern recognition receptor (RAGE) is a counterreceptor for leukocyte integrins: A novel pathway for inflammatory cell recruitment. J Exp Med 198(10):1507–1515. Chavakis T, et al. (2004) The junctional adhesion molecule-C promotes neutrophil transendothelial migration in vitro and in vivo. J Biol Chem 279(53):55602–55608. Fossati-Jimack L, et al. (2013) Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells. PLoS ONE 8(2):e57082. Holers VM, Rohrer B, Tomlinson S (2013) CR2-mediated targeting of complement inhibitors: Bench-to-bedside using a novel strategy for site-specific complement modulation. Adv Exp Med Biol 735:137–154. Vorup-Jensen T, Ostermeier C, Shimaoka M, Hommel U, Springer TA (2003) Structure and allosteric regulation of the alpha X beta 2 integrin I domain. Proc Natl Acad Sci USA 100(4):1873–1878. Xiong JP, Li R, Essafi M, Stehle T, Arnaout MA (2000) An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain. J Biol Chem 275(49):38762–38767. Svitel J, Balbo A, Mariuzza RA, Gonzales NR, Schuck P (2003) Combined affinity and rate constant distributions of ligand populations from experimental surface binding kinetics and equilibria. Biophys J 84(6):4062–4077. Gorshkova II, Svitel J, Razjouyan F, Schuck P (2008) Bayesian analysis of heterogeneity in the distribution of binding properties of immobilized surface sites. Langmuir 24(20):11577–11586.

PNAS | October 8, 2013 | vol. 110 | no. 41 | 16431

BIOCHEMISTRY

Methods

Supporting Information Bajic et al. 10.1073/pnas.1311261110 SI Methods Protein Expression and Purification. Complement component C3

proteolytic fragments were generated according to refs. 1 and 2 with minor modifications. The human complement receptor (CR) 3 I domain (subunit αM residues 127–321) mutant C128S/ I316G was subcloned into pETM-11. A codon-optimized C3d gene (encoding protein residues 993–1288 of human C3, mutant C1010A; GenScript USA, Inc.) was subcloned into pETM-20. Both proteins were expressed in BL21 (DE3) Escherichia coli cells. Cells harboring the recombinant plasmid were grown at 37 °C in 2× tryptone yeast medium where protein expression was induced at OD 600 ∼0.6 with 1 mM isopropyl β-D-1-thiogalactopyranoside and growth was continued at 20 °C overnight. All purification steps took place at 4 °C. Cell pellets were resuspended in 50 mM Hepes (pH 7.5), 300 mM NaCl, 30 mM imidazole, and 1 mM PMSF (binding buffer) and lysed by sonication. Cell debris were removed by centrifugation (20,000 × g for 30 min) and the supernatant was loaded on a Ni2+-charged HisTrap FF crude column (GE Healthcare). The recombinant proteins were eluted in 20 mL of 50 mM Hepes (pH 7.5), 300 mM NaCl, and 500 mM imidazole. The histidine tag was removed by tobacco etch virus (TEV) protease digestion during dialysis overnight against 2 L of 50 mM Hepes (pH 7.5), 300 mM NaCl, and 0.5 mM EDTA, and the cleavage product was recovered in the flow-through from a HisTrap column. The CR3 I domain was further purified on a 120 mL Superdex 75 size-exclusion chromatography column (GE Healthcare) eluted in 20 mM Hepes (pH 7.5) and 200 mM NaCl. Untagged C3d was loaded on a HiTrap Q FF 5-mL column (GE Healthcare) equilibrated in 50 mM Hepes (pH 7.5) and 150 mM NaCl and the protein recovered in the flow-through while contaminants bound to the resin. C3d mutants were generated using the QuikChange Lightning protocol (Agilent). Two different expression systems were used for the CR2 production: insect cells and E. coli. CR2 complement control protein (CCP) domains 1-2 expressed in E. coli was purified from inclusion bodies and refolded (3) with a few modifications as the construct was cloned into pETM-11 with an N-terminal hexahistidine tag and purified on Ni-nitrilotriacetic acid resin before refolding. CR2 CCP1-2 for insect cell expression was generated as follows. Briefly, by using overlap PCR primers, a gp67 secretion signal was inserted to the 5′ end of the CR2 pETM-11 construct and subsequently subcloned into pOET4 vector between XhoI and HindIII restriction sites. Recombinant viruses were generated in Sf9 cells by cotransfection of viral genomic DNA (Oxford Expression Technologies) with 1 μg of pOET4-CR2 transfer vector using Cellfectin (Invitrogen) according to the manufacturer’s protocol. The amount of virus for efficient overexpression of the protein was determined in small-scale tests without titering the virus. High Five insect cells (Invitrogen) were grown to 3 × 106 cells/ mL in BD BaculoGold Max-XP serum-free media (BD Biosciences) and were infected with 2% (vol/vol) of the third baculovirus passage. Supernatant was collected 7 d postinfection and purified on a Ni2+-charged HisTrap Excel column (GE Healthcare). The histidine tag was removed by TEV protease digestion during dialysis overnight against 2 L of 50 mM Hepes (pH 7.5), 300 mM NaCl, and 0.5 mM EDTA, and the cleavage product was recovered in the flow-through from a HisTrap column. Finally, CR2 expressed in either E. coli or insect cells was loaded on a C3d-Sepharose affinity column made in-house equilibrated in 50 mM Hepes (pH 7.5) and 50 mM NaCl. The protein was eluted by a linearly increasing NaCl gradient. Bajic et al. www.pnas.org/cgi/content/short/1311261110

Isothermal Titration Calorimetry and Surface Plasmon Resonance Assays. Isothermal titration calorimetry (ITC) experiments were

performed at 25 °C with the VP ITC (Microcal). Proteins were dialyzed against the ITC buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, and 1 mM MgCl2]. iC3b, recombinant C3d WT and mutants (100 μM) were injected in 15 aliquots of 2 μL over 4 s into the cell containing 10 μM CR3 I domain. All binding data were analyzed by fitting the binding isotherm to a simple independent binding-site model with Origin software provided with the ITC instrument. Surface plasmon resonance spectroscopy and data analysis were carried out essentially as described earlier (4) using a BIAcore 3000 instrument (GE Healthcare) to examine the binding of the CR3 and CR4 I domains to proteolytic C3 fragments. C3b, iC3b, C3c, C3dg, and C3d were coupled through primary amine groups to CM4 sensor chips. A reference flow cell was prepared by coupling of ethanolamine. CR3 or CR4 I domain in concentrations ranging from 250 nM to 100 μM, diluted in 10 mM Hepes (pH 7.5), 150 mM NaCl, and 1 mM MgCl2,, was injected at a flow rate of 10 μL/min over the sensor chips with a contact time of 250 s, followed by a dissociation phase of 150 s. The sensor chips were regenerated in 100 mM Hepes (pH 7.5), 1.5 M NaCl, and 50 mM EDTA. Following subtraction of the reference cell signal, the distribution of interactions was calculated from the resulting sensorgram using the EVILFIT algorithm (5, 6) as described earlier (4, 7). Pull-Down Assay. A CR3 I domain affinity column was generated by coupling 4 mg of purified CR3 I domain to 600 μL of CNBractivated Sepharose. C3d was added in the binding buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, and 10 mM MgCl2] and the column was washed with 10 column-volume wash steps using the same buffer. After that CR2 CCP 1-2 was added in the binding buffer and the wash procedure was repeated. Elution was carried out with 50 mM Hepes (pH 7.5), 1 M NaCl, and 50 mM EDTA. C3d D1247A and C3d D1154A mutants, defective in binding of CR3 and CR2, respectively, were used as controls. All of the volumes in wash and elution steps were exactly the same. Likewise, the volumes loaded on the SDS/PAGE (Fig. 5D and Fig. S7) were identical. Size-Exclusion Chromatography. Size-exclusion chromatography was performed on a 5-mL analytical column (Bio SEC 5; Agilent) equilibrated in 20 mM Hepes (pH 7.5), 150 mM NaCl, and 1 mM MgCl2. Before injection, the proteins were incubated for 15 min at room temperature in the presence of 1 mM MgCl2. For the analysis of complex formation the proteins were mixed in 1:1 and 1:1:1 molar ratio. The peak fractions were collected and analyzed on a silver-stained SDS/PAGE gel. The theoretical estimations of the hydrodynamic radii were done with the HydroPRO program (8) using the corresponding crystal structures. Crystallization and Data Collection. Crystallization was induced by vapor diffusion at 4 °C. Before crystallization, the CR3 I domain and C3d (both at 150 μM) were mixed in 1:1 molar ratio, added 1 mM MgCl2 and incubated at room temperature for 30 min. Crystals were grown in a few days by mixing the preincubated protein complex sample (1 μL) with an equal volume of reservoir solution [100 mM Hepes (pH 7), 19% (wt/vol) PEG 3350, 30 mM MgCl2, 5 mM NiCl2, and 2 mM dimethyloctylphosphine oxide]. Before data collection, the crystals were cryoprotected in mother liquor containing additional 30% (vol/vol) glycerol and flash-frozen in liquid nitrogen. Native data were collected at 0.9 1 of 11

Structure Determination and Analysis. The structure was solved with molecular replacement in PHASER (10) using the CR3 I

domain (Protein Data Bank ID code 1IDO) and C3d (Protein Data Bank ID code 1C3D) as search models. Rebuilding with Coot (11) and refinement with phenix.refine (12) were carried out in an iterative manner, and model quality was assessed with MolProbity (13). Buried surface area calculation was done with PISA or CNS (14, 15). All figures were prepared with the PyMOL Molecular Graphics System (v1.5.0.4; Schrödinger LLC).

1. Seya T, Nagasawa S (1985) Limited proteolysis of complement protein C3b by regulatory enzyme C3b inactivator: Isolation and characterization of a biologically active fragment, C3d,g. J Biochem 97(1):373–382. 2. Seya T, Okada M, Nishino H, Atkinson JP (1990) Regulation of proteolytic activity of complement factor I by pH: C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) have different pH optima for factor I-mediated cleavage of C3b. J Biochem 107(2):310–315. 3. White J, et al. (2004) Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli. Protein Sci 13(9):2406–2415. 4. Vorup-Jensen T (2012) Surface plasmon resonance biosensing in studies of the binding between β₂ integrin I domains and their ligands. Methods Mol Biol 757:55–71. 5. Svitel J, Balbo A, Mariuzza RA, Gonzales NR, Schuck P (2003) Combined affinity and rate constant distributions of ligand populations from experimental surface binding kinetics and equilibria. Biophys J 84(6):4062–4077. 6. Gorshkova II, Svitel J, Razjouyan F, Schuck P (2008) Bayesian analysis of heterogeneity in the distribution of binding properties of immobilized surface sites. Langmuir 24(20):11577–11586.

7. Vorup-Jensen T, et al. (2005) Exposure of acidic residues as a danger signal for recognition of fibrinogen and other macromolecules by integrin alphaXbeta2. Proc Natl Acad Sci USA 102(5):1614–1619. 8. Carrasco B, et al. (1999) Novel size-independent modeling of the dilute solution conformation of the immunoglobulin IgG Fab’ domain using SOLPRO and ELLIPS. Biophys J 77(6):2902–2910. 9. Kabsch W (2010) Xds. Acta Crystallogr D Biol Crystallogr 66(Pt 2):125–132. 10. McCoy AJ, et al. (2007) Phaser crystallographic software. J Appl Cryst 40(Pt 4):658–674. 11. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132. 12. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. 13. Davis IW, et al. (2007) MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35(Web Server issue):W375–W383. 14. Brunger AT (2007) Version 1.2 of the Crystallography and NMR system. Nat Protoc 2(11):2728–2733. 15. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372(3):774–797.

Å with a rotation of 0.1° per image on the PX1 beamline, Swiss Light Source, Villigen. Anomalous diffraction data were collected at 1.45 Å with a rotation of 0.5° per image on the ID23-1 beamline, European Synchrotron Radiation Facility, Grenoble. Data reduction was carried out with XDS (9) (Table S1).

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A

B

C

D

C3g

E

F

G

CUB CUB

C3f D1288

CUB TE C3c

TE C3c

K1001 TE Q1013

C3 convertase

C3

Q1013 factor I

C3b

C3b

factor I

iC3b

plasmin

C3dg

C3d

Fig. S1. Complement C3 and its proteolytic fragments. (A) Proteolytic fragments of C3 deposited on activator surfaces are recognized differentially by cell type-specific complement receptors on host cells. In C3 and its proteolytic fragments, the thioester (TE) domain is shown in light brown, the CUB domain in yellow, the C3a moiety in red, and the remainder of C3 in gray. The reacted TE in C3b, iC3b, and C3dg is shown as a red circle. CR1 and CR2 both consist of multiple Sushi/SCR/CCP domains, whereas CR3 and CR4 are heterodimeric integrins and CRIg has two Ig domains in its ectodomain. (B) Structure of human C3 with the TE domain in brown and the CUB domain in yellow (1). In gray is shown a C3c-like moiety, which except for small regions of the CUB domain corresponds to the proteolytic C3c fragment. (C) Structure of human C3b; notice the large shift of the TE and CUB domains compared with C3 in B (2). Q1013 marks the position of thioester glutamine that is covalently bound to the complement activator surface. (D) Close-up of the CUB and TE domains in C3b with the future C3f and C3g fragments colored dark gray and blue, respectively. The view has been rotated by 180° relative to B and C. (E) Upon double factor I cleavage, C3f is released and iC3b is formed. This is still connected to the C3c-like moiety through a single peptide bond, but according to EM (3, 4) the C3c moiety is flexibly oriented relative to the TE domain. (F) Further factor I cleavage leads to release of C3dg. (G) Final processing by other proteases, for example plasmin, may result in the stable C3d fragment. Because atomic structures of iC3b, C3dg, and full-length C3d are unknown they are represented by the corresponding residues from the C3b structure in D.

1. 2. 3. 4.

Janssen BJ, et al. (2005) Structures of complement component C3 provide insights into the function and evolution of immunity. Nature 437(7058):505–511. Janssen BJ, Christodoulidou A, McCarthy A, Lambris JD, Gros P (2006) Structure of C3b reveals conformational changes that underlie complement activity. Nature 444(7116):213–216. Chen X, Yu Y, Mi LZ, Walz T, Springer TA (2012) Molecular basis for complement recognition by integrin αXβ2. Proc Natl Acad Sci USA 109(12):4586–4591. Alcorlo M, et al. (2011) Unique structure of iC3b resolved at a resolution of 24 Å by 3D-electron microscopy. Proc Natl Acad Sci USA 108(32):13236–13240.


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Fig. S2. Binding of the CR3 (A–E) and CR4 (F–J) I domains to C3 proteolytic fragments as indicated. For each surface, the I domains were injected in 10 concentrations ranging from 250 nM to 100 μM. Experimental data (sensorgrams) are indicated in colored lines. The data were analyzed with the EVILFIT algorithm and the resulting binding isotherms are indicated in solid black lines together with the rmsd between the model and the experimental data.


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C3c

C3dg B

1000 900 800 700

−1

CR3 I domain

RU

A

0

600 500 400

−2

300

CR4

log10 kd

200 100 0

−3

C

0

D

500 450 400 350

−1

300 250 200

−2

150 100 50

−3 −8

0

−7

−6

−5

−4

−3

−2

−1

−8

−7

−6

−5

−4

−3

−2

−1

log10 KD Fig. S3. Surface plasmon resonance (SPR) analysis of the C3 fragment binding selectivity of the CR3 (A and B) and CR4 I (C and D) domains. The CR3 or CR4 I domain, stabilized by mutagenesis in the open, ligand-binding conformation (1, 2), was injected in concentrations ranging from 250 nM to 100 μM over surfaces coupled with C3c (A and C) and C3dg (B and D). The data were analyzed with the EVILFIT algorithm. The volume of interactions, indicated with colored contours [in resonance units (RU) as shown by scale bars] was plotted as a function of the dissociation constant (10−8 M ≤ KD ≤ 10−1 M) and rate (10−3 s−1 ≤ kd ≤ 100 s−1). Red arrows indicate a population of high-affinity interactions for the CR3 I domain (Kd ∼0.4 μM) shared between iC3b and C3dg but not observed for interactions with C3c.

1. Vorup-Jensen T, Ostermeier C, Shimaoka M, Hommel U, Springer TA (2003) Structure and allosteric regulation of the alpha X beta 2 integrin I domain. Proc Natl Acad Sci USA 100(4): 1873–1878. 2. Xiong JP, Li R, Essafi M, Stehle T, Arnaout MA (2000) An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain. J Biol Chem 275(49):38762– 38767.


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A Thr 209

Thr 209

Ser 144

Ser 144

Arg1254

Asp1247

Asp1247

Arg1254 Arg208

Arg208

Asp1245

Asp1245

B α7

α7

α1

α1 open Mg2+-bound

C

closed metal-free Arg208

Leu1207

2+

Ni

Lys1244

Glu224 Glu178

Phe1246

Lys1217

Arg276 Arg1254 Asn204

D Gln 204

Gln 204 Ser 142

Ser 142 Thr 209

Thr 209

Arg208

Arg208

Asp1247 Arg1254

Asp1247 Asp1245

Arg1254

Asp1245

Fig. S4. Details of the intermolecular interface and the conformation of the CR3 I domain in the complex. (A) Stereo view of the final 2mFo-DFc electron density map contoured at 1σ. (B) Comparison of the I domain bound to C3d (purple) with unbound I domain in the open conformation (Left, purple; Protein Data Bank ID code 1IDO) and the unbound I domain in the closed conformation (Right, yellow; Protein Data Bank ID code 1IDN). (C) The intermolecular interface mapped on the I domain (Left) and C3d (Right). I-domain residues within 3.5 Å of the C3d are colored brown and C3d residues in contact with the I domain are shown in purple. (D) Stereo view of the interface between of CR3 I domain (purple) and C3d (wheat) with the metal ion-dependent adhesion site (MIDAS)-coordinated nickel ion (cyan). Selected residues involved in the C3d:I domain interaction are labeled. Dashed lines indicate putative hydrogen bonds and electrostatic interactions. Water atoms are shown as red spheres.


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G

A Asp

Asp

4M76

3HI6

H

B Asp

-COO-

1L5G

3HRZ

I

C Asp

Glu 2VDO

1T0P

D

J Asp 1DZI

1T6B

E

Glu

K Asp

Glu

3Q3G

1IDO

F Asp 1MHP

Fig. S5. The nature of the acidic ligand in MIDAS-dependent protein–protein complexes correlates with the secondary structure of the ligand protein. The MIDAS-containing protein is colored purple, and the ligand protein/peptide is colored brown. (A–G) Aspartates in loop regions or in flexible termini function as MIDAS metal ion ligands. The structures are (A) the I domain:C3d complex, (B) the βI domain within the integrin α5β3 in complex with a cyclic RGD peptide, (C) the βI domain within the integrin αIIbβ3 in complex with the fibrinogen γ-peptide, (D) anthrax protective antigen complex with anthrax toxin CMG2 receptor, (E) antibody Fab complex with the CR3 I domain, (F) antibody Fab complex with the integrin VLA-1 I domain, (G) antibody Fab complex with the LFA-1 I domain, and (H) the flexible C-terminal carboxylate group from the cobra venom factor complex with the Von Willebrand factor A (VWA) domain of factor B. (I–K) The longer glutamate side chain is preferred if the ligating residue is in a rigid region within or next to secondary structure. (I) integrin LFA-1 complex with ICAM-3. (J) integrin α2β1 I domain complex with collagen. (K) A glutamate neighboring an α-helix in the ligand mimetic crystal packing of the CR3 I domain (1). Panels are shown in the same orientation with respect to the orientation of the MIDAS-containing domain except for K, which is rotated vertically for optimal presentation. The Protein Data Bank ID code of the structure is displayed on each panel.

1. Lee JO, Rieu P, Arnaout MA, Liddington R (1995) Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell 80(4):631–638.


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A aM-Crice_gris aM-Mus_musc aM-Rat_norv aM-Oryct_cun aM-Equus_cab aM-Felis_catus aM-Canis_lupus aM-Ailu_mela aM-Otol_gar aM-Sai_bol aM-Macaca_mul aM-Homo_sap aM-Pan_pan aM-Pan_tro aM-Loxo_afri aM-Sus_scrofa aM-Bos_taurus aM-Ovis_aries aM-Ovis_cana aX-Bos_tau aX-Oryct_cun aX-Mus_musc aX-Rat_norv aX-Loxo_afri aX-Homo_sap aX-Equu_caba aX-Ailu_mela aX-Canis_lupus

bA

h1

130

150 L R E C PQQE S D I L R E C PQQE S D I G L R C PQQE S D I L RGC PQQE S D I L R E C PQQE S D I L RECPRQDSD I L RECPRQDSD I I RECPRQDSD I L KGC PQQD S D I L QGC PQQD S D I L RGCP EQ . . D I L RGCPQEDSD I L RGCPQEDSD I L RGCPQEDSD I L RECPRQE SD I L RGC PQQE S D I L RGCP EQDSD I L R E C PQQD S D I L R E C PQQD S D I L P EC T NQE I D I LQECP K L EQD I QQE C P KQDQD I QQGC P RQDQD I LQECPRHEQD I RQECPRQEQD I LQECPRQEHD I LQECPRQEQD I LQECP KQEQD I

140 V V A A A A A A A A A A A A V A A A A A V V V L V V V V

F F F F F F F F F F F F F F F F F F F F F F F F F F F F

L L L L L L L L L L L L L L L L L L L L L L L L L L L L

I I I I I I I I I I I I I I I I I I I I I I I I I I I I

160 DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I DGSGS I

HPRD NN I D NS I D DSTD YEND I QSD NPTD NP I D NP I D NPND NPRE I PHD NPYD NPYD SLSD NRL D DPVD DPVD DPVD DQT D SFSN SSTD TYTD TPDD SSRN YFKD SPLD SPRD


170 QKMK E QKMK E QKMK E QRMK E QKMK E QKMKD QRMK E QRMK E QRMKD RQMK E QQMK D RRMK E RRMK E RRMK E QRMK E QRMK E E RMKR DRMK K DRMK K K RMK N A TMKN E KML D E KML A E R MM N A T MM N A KML S T KML N I KML N

a4 210 aM-Crice_gris aM-Mus_musc aM-Rat_norv aM-Oryct_cun aM-Equus_cab aM-Felis_catus aM-Canis_lupus aM-Ailu_mela aM-Otol_gar aM-Sai_bol aM-Macaca_mul aM-Homo_sap aM-Pan_pan aM-Pan_tro aM-Loxo_afri aM-Sus_scrofa aM-Bos_taurus aM-Ovis_aries aM-Ovis_cana aX-Bos_tau aX-Oryct_cun aX-Mus_musc aX-Rat_norv aX-Loxo_afri aX-Homo_sap aX-Equu_caba aX-Ailu_mela aX-Canis_lupus

NGK T NGR T NGR T L GR T GGR T RGR T L GR T YGR T QGR T T GW T YGR T L GR T L GR T L GR T GG T T L GR T FGR T FGR T FGR T NG L T MG T T RGY T RGS T KGL T QG F T RG L T GGY T GG L T

K K K H H H H H H H H H H H H H H H H F H Y R Y Y H H H

T T T T T T T T T T T T T T T T T T T T T T T T T T T T

230 ASG I ASG I ASG I ATG I ATG I ATG I ATG I ATG I ATG I ATG I ATA I ATG I ATG I ATG I ATG I ATG I ATG I ATG I ATG I ATG I ATA I ASA I ASA I ASA I ATA I ASA I ATA I ATA I

aM-Crice_gris aM-Mus_musc aM-Rat_norv aM-Oryct_cun aM-Equus_cab aM-Felis_catus aM-Canis_lupus aM-Ailu_mela aM-Otol_gar aM-Sai_bol aM-Macaca_mul aM-Homo_sap aM-Pan_pan aM-Pan_tro aM-Loxo_afri aM-Sus_scrofa aM-Bos_taurus aM-Ovis_aries aM-Ovis_cana aX-Bos_tau aX-Oryct_cun aX-Mus_musc aX-Rat_norv aX-Loxo_afri aX-Homo_sap aX-Equu_caba aX-Ailu_mela aX-Canis_lupus

B

C3-Mono_dome C3-Macr_euge C3-Ailu_mela C3-Bos_taur C3-Sus_scro C3-Equu_caba C3-Cric_gris C3-Mus_musc C3-Ratt_norv C3-Hete_glab C3-Cavi_porc C3-Otol_garn C3-Papi_anub C3-Maca_mula C3-Homo_sapi C3-Pan_trog C4-Mono_dome C4-Rat_norv C4-Mus_musc C4-Hete_glab C4-Sus_scro C4-Bos_taur C4-Equu_cab C4-Ailu_mela C4-Otol_garn C4-Gori_gori C4-Homo_sapi C4-Pan_trog


ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP ASKP GSVP ASEP ASMP ASSP ASKP ASKP ASKP ASKP ASKP

310 ADDH AGEH AGDH SREH ARDH SRDY PRDH ARDH PRDH PRDH PRDH PRDH PRDH PRDH SSEH SGDH PADH PADH PADH SEDH AHEH SHEY SREY SQEY SQEH SHEH SNEY SHEY


180 V S T VMEQF T KSK V S T VMEQF KKSK V S T VMDQ F QK S K V S T VMEQF T KSN V T I VMNQ F K K S K V S T VMGQ F K N S K V S T VMDQ F K N S K V S T VMD R F K N S K V S T VMEQF VKSK V S T VMEQF KKSK V S V MM E Q L K K S K V S T VMEQ L KKSK V S T VMEQ L KKSK V S T VMEQ L KKSK V S T VMEQF KNSK V S T VMGQ F QK S K V S T VMSQFQKSK V S T VMSR FQKSK V S T VMSR FQKSK V RA VMDR S KG T N VKAVMSQF PRP S VKAVMSQ L QRP S V KA VMSQ L QQS S VKAVMSQFQRP S VRAV I SQFQRP S VKAVMSQFQRP S VKAVMSQF RRPN VKAVMSQFQRP S

170 TLF TLF TLF SLF TLF TLF TLF TLF TLF TLF TLF TLF TLF TLF TLF TLF TLF TLF TLF TQF TQF TRF TRF TQF TQF TQF TQF TQF

S S S A S S S S A S S S S S S A S A A S S S S S S S S S

bC 190 L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ F L MQ F L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ Y L MQ F L MQ F L MQ F L MQ F L MQ F L MQ F L MQ F L MQ F

bD

REV RKV RKV LKV RKV RKV RKV LKV RKV RKV RKV RKV RKV RKV RKV RKV RKV RKV RKV RTV LRV KHV KHV Q I A QNV QVV QMV R I V

230 240 250 VRV L FQK ANGARENAVK VRE L F HK T NGARENAAK VRE L FQK I NGARDNAAK V T E L F HS S SGARANARK VRE L F HSRNGARKNA L K VRE L FQS S SGARE KA F K VRE L F HS S SGARENA L K VRE L F HS S SGARENA L K VRE L F NV T KGARENA L K V R E L F N A HQGAR E NA L K V R E L F N V NQGAR K NARK VRE L F N I T NGARKNA F K VRE L F N I T NGARKNA F K VRE L F N I T NGARKNA F K VRE L F HS S YGARKNA L K VRE L F HS K SGARENA L K VRE L F HS S SGARNHA I K VRE L F HS S SGARNHA L K VRE L F HS SNGARNHA L K VRE L F HS KNGARKSARK VDQL F HA S TGARKDA T K I T E L F T TQSGARQDA T K I T E L F T TQKGARKDA T K T NVMF S PQKGARENA T K VHR L F HA S YGARRDAAK I K E L F S A T RGARKDASK T NQL F S T S SGARKDASK T NE L F S A S KGARKDASK

180 SDEFRT SDEFR I SDEFRT SEEFRT SDTFQT SEDFQT SEDFQ I SEDFQT S DD FWT SEKFR I S E E FWT SEEFR I SEEFR I SEEFR I SNVFRT SEDFYT SDDFQT SDDFRT SDDFRT SN LMK T SNEFQT SDYFRV SHTFRT ANSFN I SNKFQT SNKF LV SDDFRV SNNFRV

a5 240

I LV I LV I LV VLV I LV I LV I LV I LV I LV I LV I LV I LV I LV I LV I LV I LV I M I I M I I M I I I I I L I VL I I L I I L I I L I I L I I L I I L I

V V V V V V V V V V V V V V V V V V V V V V V I V V V V


T T T T T T T T T T T T T T T T T T T T T T T T T T T T

250 260 270 DGEKYGDP L NY EDV DGEK FGDP L DY KDV DGEK FGDP L NY EDV DGEK FGD T L E Y EDV DGEK FGDR L E Y EDV DGEK FGDP L E Y KDV DGEKYGDP L DY KDV DGEK FGDP L DY KDV DGEK FGDP L K Y EDV DGEK FGDP L K Y EDV DGEK FGDP LGY EDV DGEK FGDP LGY EDV DGEK FGDP LGY EDV DGEK FGDP LGY EDV DGEKYGDP L NY EDV DGEK FGDP LGY EDV DGEKY L DP L E Y SDA DGEKY L DP L E YRDV DGEKY L DP L E YRDV DGEKY KDP L E Y KDV DGQK L DD P L GY E D V DGRKQGDN L S Y D S V DGRKEGDR L DYGDV DGQK EGD Y L D Y K H V DGKKEGDS L DY KDV DGQKQGD Y L GY DD V DGQKQGD Y L N Y E D V DGQK KGD S L GY E D V

a2 200 HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT HFT


NV ND ND SD KE NE NE NE KD KE EE KE KE KE NN ND ND ND ND NQ ND NN ND HN EE KD KD EV

210 FKKNPNPR LH FKRNPSPRSH FKRNPDPKSH FKRNPNPRA L F ANNPNPGS L FKKSPNPS L L FKKNPKPS F L FKANPNPRFL FQENPNPKS L FQDNPNPRS L FQRK PNPRS L FQNNPNPRS L FQNNPNPRS L FQNNPNPRS L FKKNPNPRF L FKRNPSPE L L FKRNPVPEF L FKRNSDPE L L FKRNSDL E L L FWT S R S SQS L FVSSTNPLQL F I STSSPLSL F I STSSPLRL F AHSSDPQA L FRRSSNPL S L FMD S S D P L G L FTDSSNPLVL FTYSSNPLAL

200 220 VN T VRQL VSP I KQL R VRP I QL VKP I RQL VRP I NQL VRP I EQL VKS I KQL VNA I I QL LRPVKQL VRP I RQL VNS I TQL VKP I TQL VKP I TQL VKP I TQL VNP I LQL VRP I RQL VGP I RQL V R P I GQ L V R P I GQ L VDP I VQL LDRVYQL L DS VRQL L D F VNQL L NGV KQL L A S VHQL LNSVSQL LDSVYQL LDSVSQL

bE 260


a3 190

a6 270

280 PEADRAGV I PEADRAGV I PEAEEAG I I PRAEREGV I PEADQEG I I PEADKEG I I PEADREG I I PEADREG I I PEADKEGV I PEADREGV I PEADREGV I PEADREGV I PEADREGV I PEADREGV I PEADREGV I PEADRKGV I PEADREK I I PEADRKG I I PEADRKG I I PEAEKAN I I P KAEAAG I I PMA E AA S I I PMA E AAG I I PRAEAAG I I PMADAAG I I PMA E AAG I I PMA E AAG I I PMA E AAG I I

RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYV RYA RYA RYA RYA RYA RYA RYA RYA RYA

177 221 221 221 221 208 208 208 212 221 219 221 221 221 211 221 221 221 221 226 223 242 222 227 222 223 223 223

290 I GVGNAF I GVGNAF I GVGNAF VGVGDA F I GVG I AF I GVGAAF I GVGDA F I GVGEAF I GVGDA F I GVGDA F I GVGDA F I GVGDA F I GVGDA F I GVGDA F I GVGVAF I GVGDA F I GVGDA F I GVGDA F I GVGDA F I GVGDAF I GVGL AF I GVGKAF I GVGQA F I GVGSAF I GVGL AF VGVGSAF VGVGLAF VGVGTAF

280

300 ANRKSRQE L D T NKPQSRRELDT HKPQSRRE LDT NS EQSRQE L N T S I EKSREELNT NSPKTREELNT NHL KNREELN I DNPKHREELNT N SMK S I QE L N T NS EKSRQE L N T RS SKSRQE L N T RS EKSRQE L N T RS EKSRQE L N T HS EKSRQE L N T FSEKKLEELN I N SWK S R E E L N T RGRKSRQE L D T NSKKSRKELDT NSKKSRKELDT QAHAAREELK I QVVSS LRELHD YNEHSKQEL KA YQAQSRQE L KD QKPQSKKEL KV QN R N SWK E L N D Q S T Q A WQ E L N D R K R H SWK E L N D Q KMQ SWK E L N D

258 302 302 302 302 289 289 289 293 302 300 302 302 302 292 302 302 302 302 307 304 323 303 308 303 304 304 304

a7 300

V V V V V V V V V V V V V V V V V V V V V V V I I I I I

160

220

bF 290

bB

150

FQVDN FQVDN FQVDN FRVNN FRVNN FRVNN FRVNN FRVNN FRVTN FQVNN FQVNN FQVNN FQVNN FQVNN FRVDN FQV TN FQVNN FQVNN FQVNN FKVDS FRVEN FSVEN FSVEN FQVEN FKVED F KVDD FKVDN FKVEN


320 EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL EAL AAL DAL DAL DAL DAL DAL DAL DVL DAL

310 KT NT NT NT KT KT KT KT KT KT KT KT KT KT KT KT KT KT KT SS RD KD KD RD KD RD RD RD


QDQL QNQL RNQL RNQL QNQL QNQL QNQL QNQL QNQL QNQL QKQL QNQL QNQL QNQL QNQL QNQL QNQL QNQL QNQL QKQL QGR L ENQL QNQL QNQL QNQL QNQL QNQL QNQL

284 328 328 328 328 315 315 315 319 328 326 328 328 328 318 328 328 328 328 333 330 349 329 334 329 330 330 330

interaction with C3d MIDAS water coordinator MIDAS metal-ion ligand

a9 1180 1190 1200 1210 DR L DV P K ADK F L S AAKDGNS . . E R L D E P K I E K F L S A A R GWQ K . . DK L EGD L L RK F L S T AKDKKR . . GK L EGDR L T K F L N T AKEKNR . . DKLDEP F LNK L LSTAKERNR . . GK L E E P L L NK F L S AA T DGNR . . GK L E E P Y L T K F L N T A T ERNR . . NK L E E P Y LGK F L N T AKDRNR . . NKLEEPY L TKF LNTAKDRNR . . DK L GGP L L V K F R S A A T E KNR . . ER L NGA T LQK F L NAA T EKNR . . GK L DGP I L DK F L K T AKDKNR . . GR L KGP L L NK F L T T AKDKNR . . GR L KGP L L NK F L T T AKDKNR . . GR L KGP L L NK F L T T AKDKNR . . GR L KGP L L NK F L T T AKDKNR . . E D AQNMAHNN I MAMAQGSGDD L E D L RN V AHN S LMAMA E E T GE N L E D L RN V AHN S LMAMA E E T GE H L RD L QE V AHNN LMVMAQE T GD K L EQ L QD I AHNN LMAMAQK I GDH L E D L RR A AHNN LMAMA K D I GD K L E N L RN I AHDN LMAMAQE SGD Y R E D L RN V AHRN LMAMAQK T GDN L E D L QN V AHNN LMAMAQE T GE S L A D L RGV AHNN LMAMAQE T GDN L V D L L GV AHNN LMAMAQE T GDN L A D L RGV AHNN LMAMAQE T GDN L

a10 1200 1220 . WQ E . . L AG . . WE E . . WE E . . WE E . . WE E . . WE E . . WE E . . WE E . . WE E . . WE E . . WE E . . WE E . . WE E . . WE D . . WE D . F WG P V Y WG S A Y WG L V Y WG S V F WG T V Y WG S V YWD S V Y WG S V Y WG S V Y WG S V Y WG S V Y WG S V

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AGSQ . N I GSQDN L GSQDK I T SQSN PSSQSN TTSPSN ASSQSN PGSQSN A S SQNN TGSQSN TGSQSN TGSQ I N

. . . . . . . . . . . . . . . . A V V V T V V A A A A A

. . . . . . . . . . . . . . . . V V V V L L V V V V V V

. . . . . . . . . . . . . . . . S S L S S S S P S S S S

. . . . . . . . . . . . . . . . P S R P P P P A P P P P

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TAS TPA PTA TPA TPA T LA TMA T LA T L T TPA TPA TPA

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AGSA PRNP PRSP PLSP PQRP PHSP PHSP PQGP PRNP PRNP PRNP PRNP

. . . . . . . . . . . . . . . . S S T A T A A S A S S S

. . . . . . . . . . . . . . . . E E E D D D D D D D D D

1210 1230 . . QGQK L Y N V E A T . . ERPASVNVEAT . . PGQK L Y S V E A T . . PNQK L YNV EA T . . PGQK L HN V E A T . . PGQK L Y N V E A T . . PGQK L Y N V E A T . . P DQQ L Y N V E A T . . PGQQ L Y N V E A T . . A GQK L Y S V E A T . . ARQK L YSVEAT . . PGKQL YNVEAT . . PGQQ L Y N V E A T . . PGQQ L Y N V E A T . . PGKQLYNVEAT . . PGKQL YNVEAT P T T L A P A LWV E T T P V PQA P A LW I E T T P V PQA P A LW I E T T P V PQA P A LW I E T T PMPQA P A LW I E T T P I PQA P A LW I E T T P V PQA P A LW I E T T P V P QA P A VW I E T T PMPQA P A LW I E T T P I PQA P A LW I E T T PMPQA P A LW I E T T PMPQA P A LW I E T T

SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL SYAL AYVL AYGL AYAL AYGL AYAL AYGL AYGL AYGL AYGL AYAL AYAL AYAL

a11 L L L L L L L L L L L L L L L L L L L L L L L L L L L L

1220 1230 1240 1250 AL L K I KDFDT I PGVVR AL L K LKDFDT I PDVVR AL L L LKDFDSAPPVVR AL L ARKDYDT T PPVVR AL L VVKDFDSVPP I VR AL L L LRDFDSVPPVVR AL L L LKDFDSVPPVVR AL L L LKDFDSVPPVVR AL L L LKDFDSVPPVVR AL L L LRDFDSVPPVAR AL L L LKDFDAVPPVVR AL L L LKDFDSVPPVVR AL LQLKDFDF VPPVVR AL LQLKDFDF VPPVVR AL LQLKDFDF VPPVVR AL LQLKDFDF VPPVVR H L L L REGKAE L ADQT A H L L L R EGKGEMAD K V A H L L L R EGKGKMAD K AA H L L LWE GK A E L A E QA A H L L I R EGKA EMADQ T A H L L LWE GK A E L A DQA A H L L LWE GK A EMA DQA A H L L L REGKS E EADHAA H L L L R EGKA EMADQAA H L L L H EGKA EMADQAA H L L L H EGKA EMADQA S H L L L H EGKA EMADQAA

1243 1244 1255 1253 1252 1253 1255 1254 1254 1233 1259 1255 1254 1254 1254 1254 1288 1281 1282 1285 1283 1284 1286 1284 1286 1286 1286 1286

interaction with I domain

Fig. S6. Sequence conservation of the molecular interface in the C3d:I domain complex in mammalians with α-helices and β-strands shown above each alignment. Residue numbering is shown for the human sequence with the mature numbering on top and the prenumbering below. Numbers at the ends of each sequence follow the prenumbering scheme. (A) The αM subunit of CR3. The entire I domain and flanking regions are shown. Residues in contact with C3d are marked with red triangles, those directly coordinating the MIDAS ion with arrows, and residues with hydrogen bonds to MIDAS coordinating water molecules are shown with red circles. (B) Part of C3d and a C-terminal flanking region. Residues in contact with the αM I domain are indicated with red triangles. The corresponding residues in C4 are aligned below for comparison. The figure was prepared with ALINE (1). 1. Bond CS, Schüttelkopf AW (2009) ALINE: A WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr D Biol Crystallogr 65(Pt 5):510–512.


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A Input Mw proteins kDa FT C3d CR2

50 40 30 25 20 15

C3d D1247A

C3d D1154A

FT W Elute

FT W FT W Elute

FT W FT W Elute

- + -

+ - -

+ - -

C3d WT W

10 + - -

C3d CR2

- + -

- + -

B Input Mw C3d WT proteins kDa C3d CR2 FT W Elute FT W 50

C3d D1247A

C3d D1154A

FT W FT W Elute FT W FT W Elute

40 30 25 20 15 10 C3d CR2

+ - -

- + -

+ - -

- + -

+ - -

- + -

Fig. S7. Silver-stained polyacrylamide gel of the pull-down experiments performed with the CR2 expressed in bacteria (A) and insect cells (B). A is identical to Fig. 5D. Labels + and − indicate, respectively, the presence or the absence of the protein in the pull-down assay at that particular step. From left to right, molecular weight (Mw) marker together with purified CR2 CCP 1-2 and C3d and CR3 I domain affinity column pull-down using C3d WT showing the flowthrough and wash steps and the final EDTA elution. The same samples are shown for the control experiments with the C3d mutants D1247A and D1154A. All the volumes in wash and elution steps were the same. Likewise, the volumes loaded for SDS/PAGE analysis were identical.


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V0

A

γ-glob myoglob Vtot

thyroglob

oval

vit. B12

3.87 ml

CR2:C3d

CR2

RH = 2.09 nm

RH = 1.73 nm 10

OD 280nm (AU)

10

20

30

3.50 ml

D

40

50

20

30

40

50

60

60

E

B

3.31 ml 3.47 ml

CR3:C3d

CR3 3.55 ml 10

20

30

C

40

RH = 2.74 nm

RH = 1.55 nm 50

60

10

20

F

3.48 ml

30

40

CR2:C3d:CR3 RH = 2.77 nm

RH = 1.82 nm 20

30

40

50

60

60

3.26 ml 3.45 ml

C3d 10

50

10

20

30

40

50

60

elution fractions

Fig. S8. Representative elution profiles from analytical size exclusion chromatography of CR2 (A), CR3 (B), C3d (C), and the corresponding binary (D and E) and ternary (F) complexes. The corresponding elution volumes of every peak are indicated. The column was calibrated with thyroglobulin (thyroglob, 670 kDa), γ-globulin (γ-glob, 158 kDa), ovalbumin (oval, 44 kDa), myoglobin (myoglob, 17 kDa), and vitamin B12 (vit. B12, 1.35 kDa). The void volume (V0) and total bed volume (Vtot) as well as the hydrodynamic radii calculated with HydroPRO program (1) are indicated. The binary CR2:C3d complex elutes almost at the same volume as C3d alone (D). This can be explained by the fact that their theoretical hydrodynamic radii are similar (1.82 nm for C3d and 2.09 nm for CR2:C3d). The CR3:C3d complex elutes as a double peak (E), suggesting that association/dissociation equilibrium is taking place, consistent with very fast on and off rates observed in our SPR analyses. Finally, the ternary CR2:C3d:CR3 complex elutes also as a double peak but slightly earlier than the binary CR3:C3d complex (F). The very close elution volumes can be explained by very similar hydrodynamic radii of 2.74 and 2.77 nm for the CR3:C3d and the CR2:C3d:CR3 complex, respectively.

1. Carrasco B, et al. (1999) Novel size-independent modeling of the dilute solution conformation of the immunoglobulin IgG Fab’ domain using SOLPRO and ELLIPS. Biophys J 77(6): 2902–2910.


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Table S1.

Data collection and refinement statistics

Dataset Data collection Space group Cell dimensions a, b, c, Å α, β, γ, ° Resolution, Å Rsym I/σI Completeness, % Redundancy Refinement Resolution, Å No. of reflections Rwork/Rfree No. of atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water Rmsd Bond lengths, Å Bond angles, °

Native

Anomalous

C2

C2

144.46, 65.12, 62.54 90, 115.69, 90 45.1 (2.8)* 4.9 (29.1) 16.8 (4.5) 98.1 (94.3) 3.3 (3.2)

143.40, 64.83, 62.39 90, 115.44, 90 44.9 (2.8) 6.6 (63.1) 18.4 (2.2) 97.6 (94) 3.9 (3.8)

2.8 12,885 0.1938/0.2420 3,770 2 33 74.6 53.5 44.3 0.0027 0.6594

*Values in parentheses are for the highest-resolution shell.

Table S2. Interface properties of complexes between I domains and ligands Integrin receptor Ligand Reference Interface area, Å2 P value Shape complementarity

CR3 I domain

LFA-1 I domain

LFA-1 I domain

LFA-1 I domain

VLA-2 I domain

C3d This study 491 0.54 0.71

ICAM-1 (1) 609 0.27 0.73

ICAM-3 (2) 601 0.31 0.74

ICAM-5 (3) 758 0.27 0.74

Collagen peptide (4) 609 0.59 0.76

The interface areas and the P values were calculated with PISA (5). For protein–protein interfaces P value < 0.5 means that the interface is more hydrophobic than could be expected if surface atoms were picked randomly, and P > 0.5 means that the interface is more hydrophilic than could be expected. The I domain: ligand interfaces are dominated by the electrostatic interaction between the ligand aspartate/glutamate and the MIDAS ion; however, the P values calculated from these complexes are not directly comparable to protein–protein interfaces devoid of such an interaction. The shape complementarity was calculated with the CCP4 program SC (6).

1. Shimaoka M, et al. (2003) Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112(1):99–111. 2. Song G, et al. (2005) An atomic resolution view of ICAM recognition in a complex between the binding domains of ICAM-3 and integrin alphaLbeta2. Proc Natl Acad Sci USA 102(9): 3366–3371. 3. Zhang H, et al. (2008) An unusual allosteric mobility of the C-terminal helix of a high-affinity alphaL integrin I domain variant bound to ICAM-5. Mol Cell 31(3):432–437. 4. Emsley J, Knight CG, Farndale RW, Barnes MJ, Liddington RC (2000) Structural basis of collagen recognition by integrin alpha2beta1. Cell 101(1):47–56. 5. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372(3):774–797. 6. Lawrence MC, Colman PM (1993) Shape complementarity at protein/protein interfaces. J Mol Biol 234(4):946–950.


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