1996), and citron-kinase have catalytic domains homol-. Introduction ogous to that of myotonic dystrophy kinase. PKN and its homologs have unique N-terminal ...
Molecular Cell, Vol. 4, 793–803, November, 1999, Copyright 1999 by Cell Press
The Structural Basis of Rho Effector Recognition Revealed by the Crystal Structure of Human RhoA Complexed with the Effector Domain of PKN/PRK1 Ryoko Maesaki,* Kentaro Ihara,* Toshiyuki Shimizu,* Shinya Kuroda,†‡ Kozo Kaibuchi,† and Toshio Hakoshima*§ * Division of Structural Biology † Division of Signal Transduction Nara Institute of Science and Technology 8916-5 Takayama, Ikoma Nara 630-0101 ‡ Inheritance and Variation Group, PRESTO Japan Science and Technology Kyoto 619-0237 Japan
Summary The small G protein Rho has emerged as a key regulator of cellular events involving cytoskeletal reorganization. Here we report the 2.2 A˚ crystal structure of RhoA bound to an effector domain of protein kinase PKN/ PRK1. The structure reveals the antiparallel coiledcoil finger (ACC finger) fold of the effector domain that binds to the Rho specificity-determining regions containing switch I, b strands B2 and B3, and the C-terminal a helix A5, predominantly by specific hydrogen bonds. The ACC finger fold is distinct from those for other small G proteins and provides evidence for the diverse ways of effector recognition. Sequence analysis based on the structure suggests that the ACC finger fold is widespread in Rho effector proteins. Introduction Understanding the molecular mechanisms by which the Rho family members, Rho, Rac, and Cdc42, control a variety of cellular events is a major goal of contemporary cell biology. It was initially discovered that these Rho family members control actin organization in cytoskeleton regulation, which defines cell shape, but it has become apparent that they are also involved in the regulation of cell adhesion, cell division, phagocytosis, and MAP kinase cascades and play a role in neurite outgrowth/retraction, hypertension, Alzheimer’s disease, and cellular development (Van Aelst and D’SouzaSchorey, 1997; Hall, 1998; Kaibuchi et al., 1999). Like other small G proteins, the Rho family members function as molecular switches, cycling between inactive GDPbound and active GTP-bound forms, and exert related but distinct cellular functions. For instance, the cell morphological effects induced by these members are clearly different in appearance (Ridley and Hall, 1992; Ridley et al., 1992; Kozma et al., 1995; Nobes and Hall, 1995). These differing effects are due to the different downstream effectors for these G proteins. Thus, the molecular interactions that underlie the effector binding represent key events in defining their cellular functions. Rac § To whom correspondence should be addressed (e-mail: hakosima@
bs.aist-nara.ac.jp).
and Cdc42 share a significant homology with z68% identities and, actually, bind to some common effector proteins for activation. Rho, however, exhibits relatively little similarity to both Rac and Cdc42 (z45% identities). These differences in similarity are thought to be essential for the specific activation of several downstream effector proteins by each small G protein, although we do not yet understand the molecular mechanism defining the specificity. Rho has three mammalian isoforms, RhoA, RhoB, and RhoC, that exhibit high sequence homology with 83% identities. At least ten candidate effector proteins for Rho have been identified so far. Among them, three protein serine/threonine kinases, PKN (Amano et al., 1996; Watanabe et al., 1996), Rho-kinase (Matsui et al., 1996), and citron-kinase (Madaule et al., 1998), have recently attracted a lot of attention. PKN, which is also referred to as PRK1 (Palmer et al., 1995), has a catalytic domain highly homologous to protein kinase C (Nishizuka, 1995) and phosphorylates actin cross-linking protein, a-actinin (Mukai et al., 1997), and other proteins of intermediate filaments, a second major component of the cytoskeleton. Recent biochemical studies have shown that PKN plays a specific role in the pathology of Alzheimer’s disease (Kawamata et al., 1998). Moreover, Drosophila PKN has been found to be required for dorsal closure during embryogenesis (Lu and Settleman, 1999). Rho-kinase, which is also identified as ROKa/ROCK-II and has isoforms of p160ROCK/ROCK-I/ROKb (Leung et al., 1995, 1996; Ishizaki et al., 1996; Nakagawa et al., 1996), and citron-kinase have catalytic domains homologous to that of myotonic dystrophy kinase. PKN and its homologs have unique N-terminal regulatory regions that contain a tandem repeat of three leucine zipper–like sequences with z25% identities and a basic region adjacent to the first repeat (Figure 1A). The basic region plus the first repeat are sufficient for binding to RhoA in a GTP-dependent manner (Shibata et al., 1996) whereas recent in vitro experiments (Flynn et al., 1998) have indicated that the second repeat also binds to RhoA, suggesting the existence of independent contact regions on RhoA. These unexpected multiple interactions remain largely uncharacterized. The PKN effector domain exhibits sequence homology with those of rhophilin (Watanabe et al., 1996) and rhotekin (Reid et al., 1996), but no apparent homology with those of other Rho effector proteins. Rho-kinase and its homologs are thought to make up another class of the Rho effector domains that have putative coiled-coil motifs located at the C terminus of the long coiled-coil region similar to a myosin rod. This coiled-coil type of effector domain is found in citron-kinase and an anchoring protein of kinesin motor, kinectin (Hotta et al., 1996). Moreover, another Rho effector protein, p140mDia (Watanabe et al., 1997), which belongs to a family of formin-related proteins and is highly homologous to a yeast Rho effector protein Bni1p (Kohno et al., 1996), displays another type of effector domain. No sequence homology exists between
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Figure 1. Domains of PKN and an Electron Density of the RhoA/PKN Complex (A) A diagram of PKN domains. The N-terminal basic region (blue), three leucine zipper–like motifs (green), and the catalytic domain are indicated. The three repeated regions correspond to ACC-1, -2, and -3 in Figure 4, respectively. (B) Representative 2Fo 2 Fc electron density of the RhoA/PKN complex. The map shows RhoA Lys-27 of switch I hydrogen bonded to PKN Asp-85PKN, contoured at 1.0 s with the refined model, which is indicated by white (PKN) and yellow (RhoA) carbon traces with oxygen and nitrogen atoms in red and blue, respectively.
these Rho effector domains and the Cdc42/Rac-interactive binding (CRIB) motifs of Cdc42/Rac effector proteins (Burbelo et al., 1995). To understand the diverse molecular recognition and regulatory functions of Rho, we have determined the crystal structure of the complex between the N-terminal effector domain of human PKN and dominantly active human RhoA by substituting Gly14 with valine (RhoAV14, hereafter referred to as RhoA) bound to a GTP analog, GTPgS. Results and Discussion Complex Formation and Structure Determination The N-terminal residues 33–111 of the PKN regulatory domain are sufficient for binding to RhoA in a GTPdependent manner. For our structural studies, a larger fragment containing residues 7–155 (Amano et al., 1996) was chosen to maximize protein stability. The PKN fragment forms a stable tight complex with the GTPgSbound form of dominantly active RhoA, as well as with the GTP-bound form of wild-type RhoA. The resulting complex was stable at a pH range of 6–8 and was purified by gel filtration. Crystals of dominantly active RhoA bound to GTPgS and Mg21 complexed with the N-terminal effector domain of PKN (in the following, referred to as RhoA/PKN complex) were obtained by the microbath method and were flash frozen in liquid nitrogen for data collection. Crystals of the complex contain 1:1 stoichiometry as determined by SDS-PAGE and time-of-flighttype mass spectroscopy. The structure was determined by a molecular replacement method using the uncomplexed form (Ihara et al., 1998) of GTPgS/Mg21-bound RhoA as the search model, and the molecular models were refined against the data to 2.2 A˚ resolution (Figure
1B and Table 1). The final model includes 86 residues of the PKN effector domain (spanning residues 13–98), RhoA (residues 1–181), one GTPgS molecule, one magnesium ion, and 115 water molecules. RhoA in the present complex (Figure 2) has no significant structural change as compared to that of the uncomplexed form, with a root-mean-square (rms) deviation of 0.45 A˚ for 176 Ca-carbon atoms and 0.21 A˚ for the GTPgS molecule and the Mg21 ion. The structure of the PKN effector domain is described, followed by a description of the RhoA/PKN interface that involves several specific interactions. We have found that the symmetry-related molecule of the PKN effector domain contacts RhoA in the crystal. We then describe this second interface (contact 2), which is largely hydrophobic. Subscripts such as Lys-53PKN are used to identify PKN residues. The Structure of the PKN Effector Domain The current model of the effector domain consisting of 86 residues displays a well-defined structure, while the C-terminal 57 residues and a short N-terminal segment are unstructured. The structure of the PKN effector domain features the N-terminal loop containing a short a helix (a1) and two long a helices (a2 and a3) forming an antiparallel coiled-coil fold, hereafter referred to as the ACC finger domain (Figure 2). The two long helices encompass the basic region and the first characteristic leucine repeat region, which were previously identified as the Rho-binding region. A segment of 20 residues followed by helix a3 contains a proline/glycine-rich region, suggesting that this region is highly flexible. The N-terminal region of the ACC finger domain is folded back onto the bundled helices a2 and a3 and
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Figure 2. RhoA/PKN Complex Structure (A) A ribbon representation of the PKN effector domain (blue) bound to RhoA (brown). The bound GTPgS molecule (black) and the magnesium ion (yellow) are shown in ball-and-stick models. The RhoDRs are labeled and highlighted with each color; switch I in red, strands B2 and B3 in purple, and helix A5 in orange. Switch II is colored in light green. (B) The complex is shown with RhoA depicted as a molecular surface that is colored using a gradient; bright orange indicates atoms ,4 A˚, and white atoms .7 A˚ from the bound PKN effector domain (shown as a blue tube), while lighter shades of orange indicate intermediate distances.
forms a hydrophobic core that contributes to the stabilization of the domain structure (Figure 3A). The two long helices intertwine with a slight left-handed twist. The mean angle of the axes of helices a2 and a3 is 198, and the averaged interhelical distance is 10.6 A˚. This architecture is classified as a long a-helical hairpin fold of the two-helix bundle in the SCOP protein structure database (Murzin et al., 1995). Structural comparisons with the DALI database (Holm and Sander, 1993) show that the ACC finger domain has an overall structure similar to many coiled-coil segments of functionally unrelated proteins, including the coiled-coil domains of
GreA transcript cleavage factor (Stebbins et al., 1995) and seryl-tRNA synthetase (Cusack et al., 1990; Biou et al., 1994), but is distinct from those of the recently determined Cdc42-bound CRIB motifs of WASP (AbdulManan et al., 1999) and ACK (Mott et al., 1999), which are in largely extended conformations. The ACC finger structure is also distinct from those of the Ras effector domains of c-Raf1 (Nassar et al., 1995; Emerson et al., 1995) and RalGDS (Geyer et al., 1997; Huang et al., 1997), the Ran effector domain of karyopherin-b2/importin b (Chook and Blobel, 1999; Vetter et al., 1999), and the catalytic domains of adenylyl cyclase, which bound to
Figure 3. Structure of the PKN ACC Finger Domain (A) The interhelical interactions between the two long a helices of the PKN ACC finger. Residues forming the interhelical hydrophobic core (bonds indicated in brown) and interhelical hydrogen bonds (black solid lines) are shown in ball-and-stick models (gray bonds with underlined labels). The hydrophobic interactions involving the N-terminal loop are also shown. (B) An axial helical projection looking down the helix bundle from the N and C termini of helices a2 and a3, respectively. The residues shown in blue participate in the interactions with RhoA at the interface of the RhoA/PKN complex shown in Figure 2 (contact 1). The residues participating in the second contact site (contact 2, shown in Figure 7) are in orange. Two residues, Asn-58PKN and Lys-51PKN, participate in both contacts. The leucine repeats are boxed.
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Table 1. Crystallographic Data and Phasing/Refinement Statistics Intensity Data Processing Resolution Rmergea Number of measurementsa Number of independent reflectionsa Completeness Mean ,I/s(I ).
2.2 A˚ 9.0% (31.2%)b 696,854 17,568 96% (86%)b 24.0 (2.8)b
Molecular Replacement Statistics Resolution range Rotation (a, b, g)c Translation (x, y, z)c Correlation coefficientd Rcrystg
15.0 A˚–3.0 A˚ 12.658, 42.128, 81.498 0.0477, 0.3589, 0.2987 0.493 [0.382]e, 0.673f 51.4% [55.5%]e, 44.2%f
Refinement Statistics Resolution Rcrystg Rfreeg Mean B value of RhoA Mean B value of PKN Rms bond lengths Rms bond angles Rms dihedral angles RmsDvh
2.2 A˚ 21.4% (31.5%)b 26.8% (35.9%)b 31.7 A˚2 39.6 A˚2 0.006 A˚ 1.2178 22.458 1.1798
Rmerge 5 100 3 S | I(h) 2 ,I(h). |/S I(h), where ,I(h). is the mean intensity for reflection h. The diffraction data were collected over a rotation range of 3008 with one crystal to get a higher redundancy of measurements. b Brackets are quantities calculated in the highest resolution bin at 2.28–2.20 A˚. c Eulerian angles (a, b, g) and fractional coordinates (x, y, z) are as defined in AMoRe. d Correlation coefficient is defined as S (| Fo(h) |2 2 ,| Fo (h) |2.) (| Fc (h) |2 2 ,| Fc (h) |2.) / [(S | Fo (h) |2 2 ,| Fo (h) |2.)2 S (| Fc (h) |2 2 ,| Fc (h) |2.)2]1/2, where Fo (h) and Fc (h) are observed and calculated structure factors, respectively, and ,| Fo (h) |2. and ,| Fc (h) |2. are the means of the squared observed and calculated structure factors, respectively. e Brackets are quantities of the second-best solution. f The values improved by a rigid body fitting. g Rcryst 5 100 3 S | Fo (h) 2 Fc (h) | / S Fo (h). Rfree is Rcryst which was calculated using 5% of the data, chosen randomly, and omitted from the subsequent structure refinement. h Dv is the deviation of the peptide torsion angle from 1808. a
heterotrimeric G proteins (Tesmer et al., 1997). In contrast, the a-helical structure of the ACC finger domain is reminiscent of the Rab effector domain of rabphilin3A bound to Rab3A (Ostermeier and Brunger, 1999). There is no bulged-out area or apparent kink in the bundled helices. The mean main chain dihedral angles are 266.68 for φ and 238.38 for c, as in globular proteins (Blundell et al., 1983), yet the hydrophobic interactions between the two helices are somewhat irregular. Notably, the characteristic heptad repeat, (abcdefg)n, with hydrophobic residues at the a and d positions, is broken at the middle of each of the helices a2 and a3. Instead, hydrophilic residues form interhelical hydrogen bonding interactions on the molecular surface (Figure 3A). Interestingly, one of the apparent leucine repeats (at positions 52, 59, and 66) located on helix a2 is away from the interhelical interface, while another leucine repeat of helix a3 participates in the interhelical interactions (Figure 3B).
These projected leucines, away from the interhelical interface, form a hydrophobic patch with other hydrophobic residues (Ala-62PKN from helix a2 and Pro-72PKN and Leu-76PKN from helix a3) on the molecular surface. The Interface between RhoA and the PKN ACC Finger Domain At the interface of the RhoA/PKN complex, the interacting PKN residues are clustered into two regions: box A at helix a2 and box B at helix a3 as shown in Figure 4 (PKN(ACC-1)). The interacting PKN residues are located on the molecular surface opposite that of the hydrophobic patch as described (Figure 3B). The ACC finger domain binds to RhoA at four contact sites (RhoA/ PKN contact 1 shown in Figure 5). The regions of RhoA that interact most closely with the ACC finger domain include the antiparallel b sheet B2/B3 and the N-terminal half of helix A5 located at the C terminus of the protein as well as at two ends of switch I. The interface between RhoA and the ACC finger domain is predominantly hydrophilic, and 2080 A˚2 of the accessible surface areas in the two proteins is buried in the complex (Figure 2B). The size of the buried interface is larger than those in the Rap1A/Raf-1 (Nassar et al., 1995) and H-Ras/RalGDS complexes (Huang et al., 1998). These complexes have been mediated mainly by an intermolecular antiparallel b–b interaction, which is not observed in the current RhoA/PKN complex. The major interaction between RhoA and the ACC finger domain occurs primarily at the side chain level but also includes the backbone level. An extensive hydrogen bond and salt bridge network contains 17 direct hydrogen bonds and nine water-mediated hydrogen bonds (Figure 6). At the interface, 17 residues from RhoA and 15 residues from the ACC finger domain play roles in the intermolecular interactions. Among them, the side chains of nine residues from RhoA participate in the direct hydrogen bonding interactions. The main chains of two residues (Phe-25 and Ser-26) at the switch I N terminus are directly hydrogen bonded to the ACC finger domain. Moreover, complementary electrostatic potential distribution exists at the interface, where the positively charged ACC finger domain contacts RhoA in the negatively charged region. At the heart of the interface, the ACC finger domain sticks Lys-53PKN into the shallow groove of RhoA to form hydrogen bonds with Asp-28 and the main chain of Phe-25, which is located at the base of the interface. In addition, three hydrophobic residues, Ile-46PKN, Leu-50PKN, and Leu-84PKN, contact several RhoA residues. These interactions are unlike those seen in Cdc42/WASP and Cdc42/ACK complexes, where the CRIB motifs wrap around the G protein, and in other small G protein–effector complexes including Rab3A and Ran. There is no interaction between the ACC finger domain and the RhoA extrahelical domain (Figure 2A) that defines the Rho family members. Similarly, no such interaction has been observed between the Cdc42 extrahelical domain and the CRIB motifs of WASP and ACK. The ACC finger domain has no contact with RhoA Asn-41, which is known to be ADP ribosylated by Clostridium botulinum C3 ADP-ribosyltransferase (Sekine et al., 1989), implying that the inactivation of Rho by the ADP ribosylation may not impair its effector binding itself.
Crystal Structure of the RhoA/PKN Complex 797
Figure 4. Sequence Alignment of the PKN ACC Finger Domain with the Related Rho Effector Domains The secondary structural elements (a1–a3) of the PKN ACC finger domain are indicated at the top. The residues that participate in interactions with RhoA at the interface of the RhoA/PKN complex shown in Figure 2 (contact 1) are indicated and are highlighted in yellow. These residues are clustered into box A and box B. The one-letter codes for these residues are in red (for acidic), blue (basic), and green (other hydrophilic residues). The a-g heptad repeats of helices a2 and a3 are displayed above the sequence in the coiled-coil domain. Hydrophobic residues at a and d positions are highlighted in purple. Residues that form the hydrophobic patch participating in the second contact site (contact 2, shown in Figure 7) are also indicated and are highlighted in light blue.
Rho Determinants of Effector Specificity The present structure reveals how RhoA recognizes its effector. The interface contains several contacts involving RhoA residues that have no homologous replacements in Rac/Cdc42 (Figure 5) nor other small G proteins. These conserved Rho-characteristic residues are
located at all three regions, switch I (Lys-27 and Gln29), b sheet B2/B3 (Glu-47, Gln-52, and Glu-54), and helix A5 (Glu-169) (Figures 6B and 6C). It is remarkable that these residues are different from the specificitydetermining residues (Asp-38, Val-42, Gly-47, and Leu174) of Cdc42 bound to the CRIB motifs of WASP and
Figure 5. Sequence Alignment of RhoA with the Related Small G Proteins Residues that participate in GTP/Mg21 binding are indicated at the top. The residues that participate in interactions with RhoA at the interface of the RhoA/PKN complex shown in Figure 2 (contact 1) are boxed by heavy lines with RhoDR labels. The secondary structural elements of RhoA are indicated at the top of the aligned sequences; the a helices (A1–A5) in green, extended b strands (B1–B6) in red, and 310 helices (H1, H2) in blue. Conserved residues are highlighted in yellow for the RhoA subfamily (RhoA, RhoB, and RhoC) and in red for the Rac/Cdc42 subfamily (Rac1, Rac2, and Cdc42). The residues that participate in interactions with RhoA at the second contact site (contact 2, shown in Figure 7) are boxed by thin lines and are indicated at the bottom of the alignment. Only segments around the contact regions are shown.
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Figure 6. Interactions between the PKN ACC Finger Domain and RhoA (A) A schematic representation of the direct hydrogen bonding (left) and water-mediated hydrogen bonding or van der Waals (right) interactions of RhoA/GTPgS/Mg21 with the PKN ACC finger domain at the interface of the RhoA/PKN complex shown Figure 2 (contact 1); acidic residues are shown in red, basic residues in blue, and hydrophobic residues in yellow. (B) The direct hydrogen bonds (black solid lines) between the PKN ACC finger domain and RhoA switch I and b sheet B2/B3. Thin lines indicate the hydrogen bonds involving main chain atoms. The ACC finger domain is shown in gray (a2) and light blue (a3), and RhoA in brown, with switch I in red and helix A5 in orange. The side chains forming the hydrogen bonds are shown in ball-and-stick models with labels in black (PKN) and in purple (RhoA). The bound GTPgS molecule (black) and the magnesium ion (yellow) are also shown in ball-and-stick models. The side chains of Tyr-42, Ile-23, and Pro-31, which form a hydrophobic core inside the switch I loop, and the side chains of switch I residues (39–42) are also shown. (C) The direct hydrogen bonds between the PKN ACC finger domain and RhoA helix A5 (orange) and b sheet B2/B3. The 1808 rotation of the complex from that shown in (B) is indicated to clarify the interactions between PKN helix a2 and RhoA helix A5.
ACK: the corresponding residues in RhoA are Glu-40, Ala-44, Asp-49, and Arg-176 (Figure 5). In addition to the side chains, the binding affinity is presumably conferred from contacts mediated by the main chains of switch I (Phe-25 and Ser-26), b sheet B2/B3 (Glu-47 and Val-53),
and helix A5 (Thr-163, Lys-164, and Arg-168). Thus, all these Rho specificity-determining regions, RhoDRs, including the canonical switch region both enhance the binding affinity and establish the specificity. Interestingly, the discrimination of Cdc42 from the other Rho
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family members by the CRIB motifs of WASP and ACK was also governed in part by contacts to helix A5. Moreover, Rab3A serves the C-terminal half of helix A5 as one of the Rab complementarity-determining regions, which contacts rabphilin-3A. Thus, the regions around helix A5 are important in allowing a subset of small G proteins to establish the specificity. The Switching Mechanism for Effector Recognition As in H-Ras, switches I and II of RhoA are the regions in which extensive structural changes involving main chain conformations are directly induced by GTP/GDP exchange. As deduced from structural comparison between GTPgS- and GDP-bound forms (Wei et al., 1997; Ihara et al., 1998), switch I of RhoA consists of residues 28–44, which include the N-terminal part of strand B2, while the switch II region is somewhat limited at residues 62–69, as compared with that of H-Ras (residues 60–72) (Milburn et al., 1990). The interactions of the ACC finger domain with switch I are localized to the N- and C-terminal regions, where the main chain displacements induced by GTP/GDP exchange are relatively small but significant. In addition, the nucleotide exchange induces drastic rearrangements of the side chain packing at the molecular surface of switch I, which strongly affect RhoA/ PKN interactions. In particular, one such effect is obvious for Tyr-42. In the GDP-bound form, this residue is projected toward the solvent region and sterically inhibits binding to the ACC finger domain. In the GTP-bound form, Tyr-42 is buried inside the hydrophobic core of the switch I loop and pushes out the long side chain of Lys-27 from the inside of the loop, leading to formation of the hydrogen bond with Asp-85PKN (Figure 6B). It is notable that extensive stabilization of the entire switch I structure is revealed by comparison of the temperature factor distributions in the present complexed and the uncomplexed GTP-bound RhoA molecules (data not shown). Similar structural stabilization is also observed at b sheet B2/B3, which is also flexible in the uncomplexed form. Multiple ACC Finger Domains of the PKN N-Terminal Region In addition to the first repeat of leucine zipper–like sequences forming the present PKN ACC finger structure (Figure 1A), ACC finger folds similar to the present structure seem to be retained in the second and third repeats of leucine zipper–like sequences (PKN(ACC-2) and PKN(ACC-3) shown in Figure 4, respectively), where the key residues for the ACC finger structure formation are largely conserved. In vitro experiments have shown that a fragment containing the ACC-2 region weakly binds to RhoA, while a fragment containing the ACC-3 region has no detectable binding activity to RhoA (Flynn et al., 1998). These differences in Rho binding of three PKN ACC finger domains may be explained in terms of the lack of the determinant residues for Rho binding. The ACC-3 region has alanine, leucine, or glycine at positions corresponding to the determinant residues, Lys51PKN, Lys-53PKN, Arg-60PKN, and Asp-85PKN, of the present ACC finger (ACC-1) domain. In the ACC-2 region, most determinant residues are conserved, although two determinant residues, Arg-47PKN and Arg-60PKN, are replaced with alanine and isoleucine, respectively. These
variational differences may endow ACC-2, but not ACC-3, with the tendency to bind weakly to RhoA. Another difference in ACC-2 and ACC-3 is that the residues forming the hydrophobic patch of ACC-1 are mostly conserved in ACC-2, but ACC-3 has charged residues (Glu-252PKN and Lys-256PKN) at the corresponding residues of the hydrophobic patch. As pointed out previously (Flynn et al., 1998), the evolution to a high-affinity effector domain may have been permitted by duplication of the ACC finger domains. Recently, multidomain interactions in effector recognition have also been suggested for the Raf-1/H-Ras complex, in which two distinct and nonhomologous domains, a ubiquitin-fold domain and an adjacent zinc finger–like domain, of Raf-1 bind to switch I and II regions of H-Ras, respectively (Drugan et al., 1996). The N-terminal regulatory region of PKN contains a peudosubstrate site that inhibits the protein kinase activity. In fact, the PKN N-terminal half fragment binds to the C-terminal half fragment that contains the catalytic domain. This binding inhibits the protein kinase activity, suggesting a masking mechanism by a possible autoinhibitory effect of the N-terminal region (Kitagawa et al., 1996). Since the first ACC finger domain overlaps the pseudosubstrate site, it is suggested that RhoA binding to the ACC finger domain could produce an unmasked and active catalytic domain of PKN. Hydrophobic Contacts between RhoA and the PKN ACC Finger Domain In the present crystal, RhoA contacts the symmetryrelated ACC finger domain (Figures 7A and 7B). The contact regions of RhoA include the C-terminal half and flanking region of switch II (residues 66–76) and adjacent parts from the strand B3 and switch I (contact 2 in Figure 5). This contact 2 interface is predominantly hydrophobic and buries accessible surface areas of 1640 A˚2, which is unexpectedly large for a nonspecific interface produced by crystal packing. The interface covers PKN residues localized on the hydrophobic patch of the finger’s tip, which is located on the molecular surface opposite that of contact 1 (Figure 3B). The contact 2 interactions are reminiscent of the long N-terminal a helix of the rabphilin-3A effector domain interacting with switch II of Rab3A. Superposition of two complexes using RhoA and Rab3A displays a partial but remarkable overlap of the PKN helix a2 and the rabphilin-3A N-terminal helix, which forms several hydrophobic contacts with a similar region involving switch II (Figure 7C). Notably, the C-terminal helix of the WASP CRIB motif also makes hydrophobic contact with switch II of Cdc42, with similar interactions having also been observed in the ACK/Cdc42 complex. This similarity may argue that contact 2 of the present structure may not be a result of crystal packing effects but may reflect the interactions between the ACC finger domain and RhoA in solution. In fact, RhoA and the present PKN effector domain form a complex with 1:2 stoichiometry in solution (Maesaki et al., 1999). This 1:2 complex formation has been shown to be sensitive to the GTPbound state of RhoA. The GTP dependency may be interpreted by the fact that part of switch I is involved in contact 2. Moreover, a stable conformation of switch
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Figure 7. The Second Contact Site in the RhoA/PKN Complex Compared with the Rab3A/Rabphilin-3A Complex (A) A ribbon representation of the symmetry-related PKN ACC finger domain (gray) at the second contact (contact 2) site of RhoA with the PKN ACC finger domain (blue) at contact 1 shown in Figure 2. Switch II and other regions for contact 2 are in dark green and green, respectively. (B) Surface electrostatic potentials of RhoA/GTPgS/Mg21 viewed from the same direction as in (A) with two bound PKN ACC finger domains at the contact 1 (blue) and contact 2 (gray) sites. Residues participating in contact 2 are in yellow. (C) Overlay of rabphilin-3A (purple) onto the current RhoA/PKN complex.
II in the GTP-bound state seems to be a prerequisite for the tight interactions at contact 2 whereas switch II is rather flexible and disordered in the GDP-bound state. Our observation regarding the two contact sites on RhoA may be in line with experimental results (Flynn et al., 1998; Zong et al., 1999) showing the multiple interactions of PKN with RhoA through two homologous effector domains, ACC-1 and ACC-2. Interestingly, ACC-2 preserves the residues of the hydrophobic patch for the possible contact 2 binding as mentioned. There are RhoA mutants that seem to support the second binding site with PKN (Sahai et al., 1998). Of particular interest, a series of mutations, F39A, F39V, and F39L, have resulted in the fact that two mutants, F39A and F39V, have been found to impair the RhoA binding to PKN whereas the F39L mutant, which has a large hydrophobic side chain of leucine similar to phenylalanine, has little effect on the binding. In the crystal, Phe-39 participates in the hydrophobic interactions at contact 2 but not at contact 1. Contact 2, which overlaps the binding site of p120 Rho-GAP (Rittinger et al., 1997), may account for that fact that PKN inhibits the action of Rho-GAP on RhoA (Shibata et al., 1996). However, further experimental tests are required to verify this suggestion of the second binding site. The ACC Finger Domains in Other Effector Proteins The present structure of the PKN ACC finger domain provides a clue for understanding the general architecture of the related Rho effector domains. Along with PKN homologs such as PRK2 (53% identity), rhotekin and rhophilin strongly preserve the key residues, indicating that their effector domains are folded into an ACC finger structure, as expected from their sequence homology, 43% and 26% identities, respectively (Figure 4). In rhotekin and rhophilin, some of the determinant residues are variant, and most of them have been observed in the present structure to be relevant to RhoA
binding. The N-terminal regions of the effector domains including helix a1 and the N-terminal one third of helix a2 are poorly conserved and may be folded into conformations different from that of the PKN ACC finger domain, suggesting the dispensability of this region. In fact, rhotekin lacks this region. Our close inspection of the sequence comparison shows that some characteristic residues at the boxes A and B are broadly conserved in the effector domains of Rho-kinase and its homologs, suggesting that these may fold into an ACC finger–like architecture. Based on the present ACC finger structure, results of series mutations on the effector domains of Rho-kinase and its homologs could be largely interpreted. For instance, replacement of Glu-1008 of p160ROCK, corresponding to Asp-85PKN, with alanine significantly reduces the RhoA binding (Fujisawa et al., 1996), and a double mutation containing Gln-1024 of ROKa (Leung et al., 1996), corresponding to Arg-78PKN, abolishes the binding. Rhokinase and its homologs no longer preserve exposed hydrophobic residues corresponding to the hydrophobic patch of the PKN ACC finger domain, although these hydrophobic residues are conserved in the rhotekin effector domain. In spite of the considerable divergence of sequence, it has not escaped our notice that two distinct Rho effector regions reported for kinectin (Hotta et al., 1996; Alberts et al., 1998) display partial limited homology. These effector domains are separated from each other by z180 residues, which is longer than the linker (z20 residues) between PKN ACC-1 and ACC-2 domains. Surprisingly, the kinectin ACC-1 region possesses the segment homologous to the PKN box B at the N-terminal side of the region homologous to the PKN box A, while the kinectin ACC-2 region has two regions homologous to boxes A and B in the same sequential manner as in PKN. Further structural analyses will provide more interesting information on whether these effector domains adapt in
Crystal Structure of the RhoA/PKN Complex 801
an ACC finger fold like PKN, and whether RhoA makes use of similar interactions. Differentiation of Effector Selection Recently, a new approach for the functional dissection of Rho family G proteins has been carried out using point mutations of a limited region (residues 39–42) of switch I (Joneson et al., 1996; Lamarche et al., 1996; Sahai et al., 1998). These residues do not participate in the direct interactions with the PKN ACC finger domain, but some of these mutated G proteins have been reported to select the effector proteins of specific signaling pathways. Interestingly, replacement of Tyr-42 of RhoA with cysteine abolishes the PKN binding. This effect on PKN binding is probably due to changes in the side chain packing of RhoA switch I involving Tyr42 together with Lys-27, which is hydrogen bonded to Asp-85PKN, as described above. On the other hand, this mutation exhibits no effect on the binding to ROCK-I, which has glutamic acid instead of Asp-85PKN, indicating the different natures of their interfaces in the RhoA/PKN and RhoA/ROCK-I complexes. Conclusions The structure presented reveals that PKN has a novel effector domain for Rho, which is distinct from the CRIB domains for Cdc42 and Rac. This PKN ACC finger domain binds RhoA at the RhoDRs containing switch I, b sheet B2/B3, and the C-terminal a helix A5. The specificity-determining residues of Rho are located at all the RhoDRs and are different from the specificity-determining residues of Cdc42. Thus, the present structure illustrates the various ways that the Rho family members of small G proteins interact with their effector proteins. We have also shown that the ACC finger domain is widely conserved in other Rho effector proteins including Rhokinase, which were thought to have an effector motif distinct from a class of Rho effector proteins containing PKN, rhotekin, and rhophilin. Sequence analysis based on the structure suggests that PKN has tandemly repeated ACC finger domains at the N-terminal regulatory region. The present structure provides a clue to the analysis of the multiple RhoA–PKN interactions in its utilization of multiple effector domains and, possibly, different binding surfaces on the G protein. Experimental Procedures Preparation and Crystallization of the Complex Purification of the dominantly active form of recombinant human RhoAV14 that is truncated at Ala-181 was carried out according to methods previously described (Ihara et al., 1998). The human PKN effector domain (residues 7–155), which contains a basic region and the first leucine zipper–like sequence (Amano et al., 1996), was expressed, purified, and cocrystallized with RhoAV14 bound to GTPgS and Mg21 as described (Maesaki et al., 1999). In brief, the effector domain was overexpressed in a GST-fused form using the E. coli DH5a cells, cleaved by human thrombin (Sigma), and purified by four steps of column chromatography including glutathione Sepharose 4B (Pharmacia Biotech). The resulting sample, used in this study, is verified with MALDI-TOF MS (JMS-ELITE, PerSeptive Inc) and N-terminal analysis (M492, Applied Biosystems). Analysis of the interaction of the two proteins by electrophoretic mobility shift assay (EMSA) and gel filtration using Sephacryl S-100 or Superose 12 (Pharmacia Biotech) indicated that RhoA and PKN(7-155) form a 1:2 complex in a GTP-dependent manner (Maesaki et al.,
1999). Complex crystals between RhoAV14/GTPgS/Mg21 and PKN(7155) were obtained at 48C from solutions containing 50 mM Bis-Tris (pH 6.5), 15 mM Ca(CH3COO)2, 15% PEG 300, and 1:2 complex purified by gel filtration. However, the crystals, which belong to space group P41212 or P43212 (a 5 b 5 66.90 A˚, c 5 149.54 A˚), were found to contain one 1:1 complex in the asymmetric unit, which was verified by SDS PAGE, MALDI-TOF MS, and EMSA. The Vm value (2.2 A˚3/Da) also suggested one 1:1 complex in the asymmetric unit. Data Collection and Structure Determination X-ray diffraction data were collected at 2.2 A˚ resolution using a Rigaku R-Axis IV on a rotating anode generator (Rigaku FR-C) with CuKa radiation at 100 K using a Rigaku Cryosystem. The focus size of the X-ray beam was 100 mm, and the beam was focused using a Super double-focusing mirror. All data were processed using the programs DENZO and SCALEPACK (Otwinowski and Minor, 1997) (Table 1). The initial phases were calculated by the molecular replacement method with the program AMoRe (Navaza, 1994) using a search model based on the human RhoAV14/GTPgS/Mg21 structure (Ihara et al., 1998). Several searches have been done using different ranges of intensity data and integration radii. Rigid body refinements of the search model performed with the program CNS (Brunger et al., 1998) resulted in a correlation coefficient of 0.669 and an Rcryst value of 44.8% in P41212. The resultant initial map shows clear electron densities for most of RhoAV14, and residual densities for the PKN effector domain, exhibiting typical features for a helices in a coiled-coil form. Structure Refinement The models were built and refined through alternating cycles using the programs O (Jones et al., 1991) and CNS, respectively. Two regions of the PKN effector domain were poorly defined in the resulting map. The first is at the six N-terminal residues, and the second is at the 60 C-terminal residues that are followed by helix a3. After several cycles of refinement, we could not define these regions that have uninterpretable densities that imply complex disorder. The final model, which was refined with a crystallographic R value of 21.4% (free R value of 26.8%) for all intensity data at 2.2 A˚ resolution, includes 86 residues of the PKN effector domain (spanning residues 13–98), RhoAV14 (residues 1–181), one GTPgS molecule, one magnesium ion, and 115 water molecules. A summary of the refinement statistics is given in Table 1. The PKN effector domain may have two rotamers of the His-88 side chain in the imidazole ring plane, which could not be determined in the current analysis. There is no residue in disallowed regions of the Ramachandran plots as defined in PROCHECK (Laskowski et al., 1993). The structure was inspected using the programs QUANTA (Molecular Simulators Inc.), GRASP (Nicholis et al., 1991), and MOLSCRIPT (Kraulis, 1991). Acknowledgments This work was supported by Grants in Aid for Scientific Research to T. H. (09308025, 10359003) and for Scientific Research on Priority Areas to T. H. and K. K. (1017903-4) from MESSC of Japan, and partly by grants from JSPS (K. K.). K. I. was supported by a research fellowship for Young Scientists from JSPS. T. H. is a member of Tsukuba Advanced Research Alliance (TARA Sakabe project) of Tsukuba University. Received July 15, 1999; revised September 2, 1999. References Abdul-Manan, N., Aghazadeh, B., Liu, G.A., Majumdar, A., Ouerfelli, O., Siminovitch, K.A., and Rosen, M.K. (1999). Structure of Cdc42 in complex with the GTPase-binding domain of the ‘Wiskott-Aldrich syndrome’ protein. Nature 399, 379–383. Alberts, A.S., Bouquin, N., Johnston, L.H., and Treisman, R. (1998). Analysis of RhoA binding proteins reveals an interaction domain conserved in heterotrimeric G protein b subunits and the yeast response regulator protein Skn7. J. Biol. Chem. 273, 8616–8622. Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996). Identification
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