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Structure of components of an intercellular channel complex in sporulating Bacillus subtilis Vladimir M. Levdikov, Elena V. Blagova, Amanda McFeat, Mark J. Fogg, Keith S. Wilson, and Anthony J. Wilkinson1 Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom

Following asymmetric cell division during spore formation in Bacillus subtilis, a forespore expressed membrane protein SpoIIQ, interacts across an intercellular space with a mother cell-expressed membrane protein, SpoIIIAH. Their interaction can serve as a molecular “ratchet” contributing to the migration of the mother cell membrane around that of the forespore in a phagocytosis-like process termed engulfment. Upon completion of engulfment, SpoIIQ and SpoIIIAH are integral components of a recently proposed intercellular channel allowing passage from the mother cell into the forespore of factors required for late gene expression in this compartment. Here we show that the extracellular domains of SpoIIQ and SpoIIIAH form a heterodimeric complex in solution. The crystal structure of this complex reveals that SpoIIQ has a LytM-like zinc-metalloprotease fold but with an incomplete zinc coordination sphere and no metal. SpoIIIAH has an α-helical subdomain and a protruding β-sheet subdomain, which mediates interactions with SpoIIQ. SpoIIIAH has sequence and structural homology to EscJ, a type III secretion system protein that forms a 24-fold symmetric ring. Superposition of the structures of SpoIIIAH and EscJ reveals that the SpoIIIAH protomer overlaps with two adjacent protomers of EscJ, allowing us to generate a dodecameric SpoIIIAH ring by using structural homology. Following this superposition, the SpoIIQ chains also form a closed dodecameric ring abutting the SpoIIIAH ring, producing an assembly surrounding a 60 Å channel. The dimensions and organization of the proposed complex suggest it is a plausible model for the extracellular component of a gap junction-like intercellular channel. sporulation

| alternate sigma factor | transcription regulation

C

ells in eukaryotic tissues frequently exchange metabolites and relay signals through clusters of intercellular channels collectively known as gap junctions. Formation of these channels occurs through docking of hexameric protein assemblies called connexons and requires close apposition of the respective cell membranes leaving a narrow (∼2 nm) gap. Bacteria are known to form multicellular communities; moreover, they can transfer molecules such as DNA during bacterial conjugation, or virulence proteins during infection, directly to the cytoplasm of recipient cells. However, these processes require the elaboration of specialized secretion machinery spanning the envelopes of the donor and recipient cells and bridging a much wider intercellular space. It is only recently that intercellular pores analogous to gap junction-type channels have been discovered in bacteria, during spore formation in Bacillus subtilis (1, 2). Sporulation is an extreme adaptation to starvation involving intimate interactions between two cells and a series of morphological changes that produces a dormant spore (3). It begins with an asymmetric cell division generating cells of unequal size that initially lie side by side (Fig. 1A). The membrane of the mother cell then migrates around that of the forespore in a phagocytosis-like process, at the completion of which there is membrane fission and the engulfed forespore is released into the mother cell cytoplasm as a cell within a cell surrounded by a double membrane. A thick layer of peptidoglycan, known as the cortex, is formed between these membranes, and a thick proteinaceous coat is deposited around the maturing forespore.

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Finally, the mother cell undergoes a process of programmed cell death, releasing the mature spore, which can survive indefinitely. The different fates of the two cells are determined by parallel programs of gene expression orchestrated by alternate RNA polymerase σ-factors whose activities are controlled by intercellular signaling pathways and linked to morphological development. Four cell-specific σ-factors are activated sequentially, in the order σF, σE, σG, and σK (3, 4), with σF and σG active in the forespore and σE and σK active in the mother cell (Fig. 1A). The earliest acting sporulation-specific sigma factor, σF, is regulated posttranslationally to ensure that σF activity is delayed pending completion of the asymmetric septum and confined to the forespore. σF activity triggers a signaling pathway leading to the activation of σE in the mother cell, and together these two sigma factors control the expression of genes required for engulfment. Upon completion of engulfment, σG activity develops in the forespore, and this in turn sets in train an intercellular signaling pathway leading to the activation of σK in the mother cell. Together, σG and σK control the expression of genes required for spore maturation and release. Relative to other σF-controlled genes, transcription of sigG (the gene encoding σG) is delayed; moreover, premature σG-directed transcription is prevented by the action of a saturable anti-σG factor, Gin (5). Realization of σG activity is coupled to the faithful completion of engulfment (6) and requires SpoIIQ, whose gene is expressed in the forespore under σF control (7), and the eight proteins encoded by the spoIIIA operon that is transcribed in the mother cell under σE control (8). SpoIIIAH and SpoIIQ colocalize, initially at the sporulation septum and subsequently along the engulfing membrane surrounding the forespore (9, 10). Each protein has a single membrane-spanning segment close to its amino terminus, through which it is attached to the cell membrane, and an extracellular domain, through which the two proteins interact across the intermembrane space (Fig. 1B). The interactions of SpoIIQ and SpoIIIAH have been proposed to function as a molecular “zipper” or “ratchet” facilitating membrane migration during engulfment (9, 11). It has recently been suggested that SpoIIIAH and SpoIIQ form a channel connecting the mother cell and the forespore (1, 2). The extracellular domain of SpoIIIAH, which is expressed in the mother cell, was shown to be accessible from the forespore cytoplasm; moreover, the C terminus of SpoIIIAH has sequence similarity to members of the YscJ/FliF family, which form multimeric rings in type III secretion systems and flagellae. This channel was originally assumed to be a conduit for the passage from the mother cell to the forespore of a specific, but putative, regulator of σG (1).

Author contributions: K.S.W. and A.J.W. designed research; V.M.L., E.V.B., A.M., and M.J.F. performed research; M.J.F. contributed new reagents/analytic tools; V.M.L., A.M., K.S.W., and A.J.W. analyzed data; and A.J.W. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: The crystallography, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3TUF). 1

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

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1120087109/-/DCSupplemental.

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Edited by Thomas J. Silhavy, Princeton University, Princeton, NJ, and approved February 17, 2012 (received for review December 9, 2011)

Fig. 1. Engulfment and σ-factor activation during spore formation in B. subtilis. (A) After asymmetric cell division, the forespore (upper part of left images) and the mother cell (lower part) lie side by side and are surrounded by peptidoglycan (gray). Peptidoglycan degradation and septal thinning is followed by migration of the mother cell membrane around that of the forespore, membrane fission, and release of the forespore into the mother cell cytoplasm. Compartment- and stage-specific σ factors are activated as indicated. (B) The domain structure of SpoIIQ (Q) and SpoIIIAH (AH) with the transmembrane (TM) segments indicated.

Subsequently, it has been proposed that the channel is a gap junction-like feeding tube through which the mother cell supplies small molecules for the biosynthetic needs of the forespore (12). Here our interest is in the structural basis of the cell–cell interaction mediated by SpoIIQ and SpoIIIAH. We have expressed and purified the extracellular domains of the two proteins and measured the stability and stoichiometry of their complex in solution. In addition, we have determined the crystal structure of the SpoIIQ–SpoIIIAH complex and used the homologous relationship between SpoIIIAH and the pore-forming type III secretion system protein EscJ, which belongs to the YscJ/FliF family, to generate a model for the SpoIIQ–SpoIIIAH components of the proposed intercellular channel. The plausibility of the model supports the proposal that the cytoplasms of the mother cell and the forespore are indeed connected by an intercellular channel. Meisner et al. have solved the structure of SpoIIQ–SpoIIIAH in a different crystal form and drawn broadly similar conclusions (13). Results Extracellular Domains of SpoIIQ and SpoIIIAH Form a Heterodimeric Complex. The extracellular domains of SpoIIQ (residues 43–283)

and SpoIIIAH (residues 32–218) were overproduced as fusion proteins with an N-terminal cleavable hexahistidine tag. The proteins were purified by steps of nickel affinity and gel filtration chromatography, with the affinity tag being removed by digestion with HRV 3C protease to leave a residual GPA tag. To determine the quaternary structures of the respective proteins and their complex, we performed size exclusion chromatography with multiangle laser light scattering (SEC-MALLS), which enabled the molecular mass of eluting species to be deduced. SpoIIQ(43–283), SpoIIIAH(32–218), and mixtures of the two proteins were analyzed (Fig. 2). For SpoIIQ(43–283) and SpoIIIAH(32–218), the traces show single peaks associated with molecular masses of 25.8 kDa and 20.8 kDa, respectively. The calculated masses of the recombinant proteins are 26.4 kDa and 21.2 kDa, showing that both SpoIIQ(43–283) and SpoIIIAH (32–218) are monomers. Following mixing, there is a pronounced shift in the principal refractive index peak to lower retention volumes and the new species has a weight-average molecular mass of 41.4 kDa. The calculated Mr of a 1:1 complex is 47.6 kDa. We 5442 | www.pnas.org/cgi/doi/10.1073/pnas.1120087109

Fig. 2. Molecular mass measured from SEC-MALLS analysis. One hundredmicroliter samples of 2 mg·mL−1 protein mixtures at the approximate molar ratios indicated were loaded onto a Superdex S200 10/300 gel filtration column. The continuous lines trace the refractive index of the eluate from this column as a function of time. The dotted lines represent the weight average molecular weight of the species in the eluate calculated by using the lightscattering measurements.

conclude that SpoIIQ(43–283) and SpoIIIAH(32–218) form a heterodimer. The equilibrium dissociation constant of the SpoIIQ (43–283)–SpoIIIAH(32–218) complex measured by surface plasmon resonance and isothermal titration calorimetry is 1 μM (Fig. S1 A and B and SI Materials and Methods), close to values obtained for complexes of different-sized fragments of these two proteins in an earlier study (14). Structure Solution. The crystal structure of the SpoIIQ–SpoIIIAH complex was solved by single-wavelength anomalous dispersion using crystals grown from SpoIIIAH and the selenomethionine derivative of SpoIIQ (Table S1). There is one molecule each of SpoIIQ (43–283) and SpoIIIAH(32–218) in the asymmetric unit (Fig. 3A). The SpoIIIAH chain can be traced from residues 103 to 217 and the SpoIIQ chain from residues 75 to 232. Residues 32 to 102 and 218 of SpoIIIAH and residues 43 to 75 and 233 to 283 of SpoIIQ, representing close to 40% of the protein content of the unit cell, are not discernible in the electron density maps and are assumed to be disordered. The circular dichroism spectra of SpoIIQ(43–283), SpoIIIAH(32–218), and their complex are consistent with a high proportion of random coil (Fig. S1C). The spectrum for the SpoIIQ (43–283)–SpoIIIAH(32–218) complex is similar to the sum of the spectra of the individual proteins indicating little or no change in secondary structure content upon complex formation. Structure of SpoIIQ. Residues 75 to 232 of SpoIIQ form 13 β-strands arranged in three β-sheets (I, β1-β4-β12-β8-β7-β6-β10; II, β9-β11β5; III, β2-β3) and a single α-helix (Fig. 3A and Fig. S2). As anticipated, the top scoring matches following a structural similarity search using the program DALI (15) are metalloproteases of the M23 family, exemplified by LytM of Staphylococcus aureus [Protein Data Bank (PDB) accession no. 2B13 (16)], which gives a Zscore of 15.4 with 122 Cα atoms superposing with a positional rmsd of 2.3 Å. The superposition is especially close across β-sheets I and II (Fig. S2C). A notable difference in the structure is the extended segment between β1 and β4 (residues 94–121), which, in SpoIIQ, contains the α-helix as well as a β-turn formed by strands β2 and β3 in a protrusion from the core of the protein that forms the SpoIIIAH interface. The M23 metalloproteases contain a conserved quintet of two histidines and an aspartate, which coordinate a zinc atom, and two Levdikov et al.

(Fig. 3A and Fig. S3A). Helices α1 and α2, containing six and eight turns, respectively, run in antiparallel directions and form extensive interactions with each other. The plane of the β-sheet (strand order β1-β2-β3 with β1 running antiparallel to the other two strands) is perpendicular to that formed by helices α1 and α2, and the edge of β1 packs between these helices as they splay apart. Three arginine side chains from α1 (Arg110, Arg117, and Arg121) play prominent roles in organizing the structure in ion-pairing networks involving Glu114, Glu152, Glu159, Asp169, and Glu175, with Asn173 also implicated. Numerous apolar side chains project from the distal face of the β-sheet (Fig. 3A) to form packing interactions with helices α2, α3, and α4. The proximal face of this sheet is noticeably more polar and exposed. A Dali database search for proteins with structural similarity to SpoIIIAH (15) confirmed the close relationship to the YscJ/FliF family (PDB accession no. 1YJ7) inferred from sequence comparisons, with EscJ as the highest scoring hit (Z-score = 8.7), with 82 equivalent Cα atoms superposing with an rmsd of 2.4 Å (Fig. 4A). The matching residues are in the C-terminal domain of EscJ and a segment of SpoIIIAH extending across helix α2 and the β-sheet to the C terminus of the protein (Fig. 4A and Fig. S3B). This corresponds well to the region of sequence homology identified previously (1, 2). The long α1 helix in SpoIIIAH is replaced in EscJ by an extended, partially disordered segment that links to the N-terminal domain (Fig. 4A).

histidines that participate in acid–base catalysis (16). Two of the metal-coordinating residues, Asp123 and His204, together with His202, are conserved in SpoIIQ and these residues superpose closely in the structures (Fig. S2 B–D). The other two histidines, including one that coordinates the metal, are replaced by serines and zinc is not present in SpoIIQ. This observation is consistent with mutagenesis experiments in which alanine substitution of His202, Asp123, and His204 has no effect on sporulation (12). As shown in Fig. S2E, the prominent substrate binding cleft leading to the metal-coordination site in LytM is closed off in SpoIIQ by the β2-β3 hairpin. Consistent with these observations, there is no evidence that SpoIIQ is a protease. Deletion of residues 202 to 216 prevents σG activation and blocks sporulation (12). As these residues span β12 at the heart of the structure, their deletion would be expected to disrupt protein folding and lead to loss of function. The chain cannot be traced before residue 75 or past residue 232, even though residues 43 to 283 were shown to be present in dissolved crystals. The C-terminal 50 residues of SpoIIQ can be truncated without a discernible effect on the sporulation efficiency (12). These residues are very poorly conserved among SpoIIQ orthologues, have a preponderance of polar residues, and are not predicted to form a discrete structure. Structure of SpoIIIAH. The SpoIIIAH(103–217) chain consists of

four α-helices and three β-strands in the order α1-α2-β1-β2-α3-α4-β3

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Modeling of SpoIIQ–SpoIIIAH Channel. The C-terminal domain of EscJ, whose fold is shared by SpoIIIAH, has been identified as a ring-building motif that allows assembly of variably sized membrane rings in type III secretion systems (18). The asymmetric unit of the EscJ crystal contains four chains that lie side by side, interacting across their long axes (Fig. 4C) (19). Following superposition of SpoIIIAH onto one of these four EscJ chains (Fig. 4D), helix α1 of SpoIIIAH overlays—albeit in an antiparallel direction—an α-helix in the neighboring EscJ subunit. As a result, it is straightforward to superimpose two SpoIIIAH chains onto the four EscJ chains (Fig. 4E). The SpoIIIAH–SpoIIIAH interface in this modeled structure is formed by the two long helices α1 and α2. As can be deduced from Fig. 4 B and E, this interface is orthogonal to the SpoIIQ interaction surface, which is formed by α2, α4, and β3. The EscJ crystal has a sixfold crystallographic screw axis of symmetry, which generates helices of EscJ chains with 24 protomers per turn (19), although, in the functioning type III secretion system setting, these protomers form a planar 24-fold symmetric ring. Application of a sixfold symmetry operator to the pair of SpoIIIAH chains overlaid onto the four EscJ chains of the asymmetric unit generates a circular assembly of 12 SpoIIIAH chains surrounding PNAS | April 3, 2012 | vol. 109 | no. 14 | 5443

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Fig. 3. Structure of the SpoIIQ–SpoIIIAH complex. (A) Ribbon diagram of the complex formed between the extracellular fragments of SpoIIIAH (green) and SpoIIQ (light blue). (B) Stereo diagram of the interface. The side chains of residues forming significant intermolecular contacts are shown as cylinders, as are the main chain atoms of residues in the intermolecular β-sheet. Atoms are colored by element with carbons colored by chain as in A. Polar interactions are represented as dashed lines.

Protein–Protein Interface. The SpoIIQ and SpoIIIAH molecules in the asymmetric unit of the crystal form a heterodimer. The protein–protein interface is formed by the protruding helix–β-turn element in SpoIIQ and the α/β subdomain of SpoIIIAH. At the heart of the molecular interface, β3 from SpoIIIAH aligns with β3 from SpoIIQ to create a five-stranded intermolecular β-sheet (Fig. 3). Meanwhile, helix α3 in SpoIIIAH packs across α1 in SpoIIQ. In the protein–protein interface, 1,550 Å2 of surface area is buried. This is sufficient for stable and specific association of two proteins and is in the range of buried surface areas observed for nonobligate protein–protein complexes (17). The SpoIIQ binding surface of SpoIIIAH is distributed across the C-terminal 30 residues with Ala193, Thr194, Ile197, Val210, Ala211, and Val212 at the core and His188, Lys190, Lys205, Asp209, Thr213, and Phe214 constituting the rim (17). For the SpoIIIAH binding surface of SpoIIQ, the core is formed by Leu109, Tyr116, and Leu118, with residues Tyr96, Thr98, Glu106, Asn114, Thr115, Ser117, Lys120, and Val212 prominent in the rim. Twelve polar interactions, including a salt-bridge, complement the largely apolar interactions among the side chains of the core residues (Fig. 3B).

Fig. 4. Comparison of SpoIIIAH and EscJ and model for the channel. (A) Ribbon representation of SpoIIIAH (green) and EscJ (magenta) following their superposition. The closest overlap is between the lower C-terminal domain of EscJ and the β-strand region of SpoIIIAH. (B) EscJ superposed on SpoIIIAH with the bound SpoIIQ (light blue), viewed orthogonal to A about the vertical axis. (C) Ribbon tracing of four molecules from the asymmetric unit of the EscJ crystal with alternate chains colored red and magenta. (D) Superposition of SpoIIIAH onto a magenta chain of EscJ as in A. The overlap of α1 from SpoIIIAH with an α-helix in the adjacent red EscJ chain is apparent. (E) Superposition of two SpoIIIAH subunits onto four subunits of EscJ. (F) Ribbon (Left) and surface (Right) representation of the modeled dodecameric SpoIIQ–SpoIIIAH assembly. The SpoIIQ chains are in light blue, with one chain in dark blue. The SpoIIIAH chains are in green, with one chain picked out in dark green. The assembly is viewed from the mother cell side (Left) and the forespore side (Right). (G) Orthogonal view of the modeled SpoIIQ–SpoIIIAH assembly with additional transmembrane helices in the context of the intercellular space and the pores in the respective membranes, which allow the passage of yet-unidentified factors required for σG activity.

a channel (Fig. 4F). The long helices α1 and α2 narrow the diameter of the channel from 75 Å in EscJ to 58 Å in the SpoIIIAH model. Application of the symmetry to the SpoIIQ–SpoIIIAH complex inevitably generates a circular dodecameric assembly of SpoIIQ, adjacent to that formed by SpoIIIAH. It would not necessarily follow that the SpoIIQ chains would form a closed ring. They do so, however, because the SpoIIQ binding surface of SpoIIIAH is orthogonal to the modeled SpoIIIAH–SpoIIIAH interface, and because the lateral dimensions of the SpoIIQ molecule match those of SpoIIIAH. It is likely that further adjustment of the interfaces of one or both subunits accompanies channel formation, nevertheless, the close fit and the paucity of steric clashes in our simple model are striking (Fig. 4F). Thus, the closely apposed rings of SpoIIQ and SpoIIIAH shown in Fig. 4F, and in the context of the cell membranes in Fig. 4G, represent a plausible model for the extracellular component of an intercellular channel. The observation of heterodimers of SpoIIQ(43–283) and SpoIIIAH(32–218) indicates that missing factors such as the transmembrane regions and/or the seven additional proteins encoded by the spoIIIA operon are required for the assembly of 5444 | www.pnas.org/cgi/doi/10.1073/pnas.1120087109

the dodecamer channel. Most of the SpoIIIA proteins have similarities to components of protein secretion systems (6), and they may interact with regions of SpoIIQ and SpoIIIAH that are disordered in the crystal. The diameter of the lumen in the modeled SpoIIIAH–SpoIIQ complex is 58 Å at its narrowest point. This is clearly wide enough to allow passage of small metabolites or larger proteins, although the width of the channel may be restricted by other components in a larger assembly, and the channel may be gated on the mother cell side with substrate translocation coupled to the ATPase activity of SpoIIIAA (6). Discussion The structure determined here allows the visualization of an intercellular interaction between two proteins essential for cell development in B. subtilis. The binding of SpoIIQ and SpoIIIAH has several consequences. First, it is responsible for the capture of SpoIIIAH at the septal membrane and the subsequent localization at this site of other protein complexes required for engulfment (9). Second, SpoIIQ binding to SpoIIIAH can support the migration of the engulfing mother cell membrane around the Levdikov et al.

1. Meisner J, Wang X, Serrano M, Henriques AO, Moran CPJ, Jr. (2008) A channel connecting the mother cell and forespore during bacterial endospore formation. Proc Natl Acad Sci USA 105:15100–15105. 2. Camp AH, Losick R (2008) A novel pathway of intercellular signalling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels. Mol Microbiol 69:402–417. 3. Hilbert DW, Piggot PJ (2004) Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol Mol Biol Rev 68:234–262. 4. Losick R, Stragier P (1992) Crisscross regulation of cell-type-specific gene expression during development in B. subtilis. Nature 355:601–604. 5. Karmazyn-Campelli C, et al. (2008) How the early sporulation sigma factor sigmaF delays the switch to late development in Bacillus subtilis. Mol Microbiol 67:1169–1180. 6. Doan T, et al. (2009) Novel secretion apparatus maintains spore integrity and developmental gene expression in Bacillus subtilis. PLoS Genet 5:e1000566. 7. Londoño-Vallejo JA, Fréhel C, Stragier P (1997) SpoIIQ, a forespore-expressed gene required for engulfment in Bacillus subtilis. Mol Microbiol 24:29–39. 8. Illing N, Errington J (1991) The spoIIIA operon of Bacillus subtilis defines a new temporal class of mother-cell-specific sporulation genes under the control of the sigma E form of RNA polymerase. Mol Microbiol 5:1927–1940. 9. Blaylock B, Jiang X, Rubio A, Moran CPJ, Jr., Pogliano K (2004) Zipper-like interaction between proteins in adjacent daughter cells mediates protein localization. Genes Dev 18:2916–2928. 10. Rubio A, Pogliano K (2004) Septal localization of forespore membrane proteins during engulfment in Bacillus subtilis. EMBO J 23:1636–1646. 11. Broder DH, Pogliano K (2006) Forespore engulfment mediated by a ratchet-like mechanism. Cell 126:917–928. 12. Camp AH, Losick R (2009) A feeding tube model for activation of a cell-specific transcription factor during sporulation in Bacillus subtilis. Genes Dev 23:1014–1024.

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interdigitate with the opposite connexon by 6 Å, producing an intercellular gap of 40 Å. The width of the SpoIIQ–SpoIIIAH complex in Fig. 4 is approximately 55 Å, placing a lower limit on the width of the intermembrane space in engulfing B. subtilis. Further work is needed to determine the full structure and composition of the intercellular channel and, most interestingly, the nature of the molecules that pass through it. Materials and Methods SpoIIQ(43–283) and SpoIIIAH(32–218) were produced in high yield as recombinant proteins in Escherichia coli and purified as described in SI Materials and Methods. Large single crystals of their complex were obtained in hanging drops composed of 1 μL of 45 mg·mL−1 complex and 1 μL of reservoir solution containing 0.1 M sodium acetate, pH 4.5, and 50% (vol/vol) polyethylene glycol 400 as described in SI Materials and Methods. X-ray diffraction data from a native crystal and a crystal containing the selenomethionine-derivatized SpoIIQ at three wavelengths were collected at the Diamond Light Source beamlines I04 and I02, respectively (Table S1 and SI Materials and Methods). Single-wavelength (0.9796 Å) data on the selenomethionine derivative crystal were sufficient for SAD structure solution. An initial model for the one heterodimer in the asymmetric unit was constructed by using Buccaneer (24). The native data were 99.3% complete to 2.75 Å resolution. Despite substantial anisotropy in the structure factor amplitudes (0.29, 0.41, 1.00), all data were used in the refinement as the Rfree factor for the final model in the highest resolution bin was reasonably low. Cycles of refinement with REFMAC (25) iterated with model building gave a final R factor of 0.165 (Rfree = 0.226) to 2.26 Å resolution. The refinement statistics are summarized in Table S1. The coordinates and structure factors have been deposited in the PDB, with accession no. 3TUF. SEC-MALLS, circular dichroism, isothermal titration calorimetry, and surface plasmon resonance experiments were carried out as described in SI Materials and Methods. ACKNOWLEDGMENTS. The authors thank Charles Moran and Christine Dunham for discussion and data sharing before publication and Johan Turkenburg, Sam Hart, Andrew Leech, Richard Jones, Jen Potts, Gideon Davies, and staff at the Diamond Light Source for help and advice. This work was supported by European Union Contracts LSHG-CT-2006-037469 (BaSysBio) and LSHG2-CT-2006-031220 (Spine2-Complexes) and by Wellcome Trust Project Grant 082829.

13. Meisner J, et al. (2012) Structure of the basal components of a bacterial transporter. Proc Natl Acad Sci USA 109:5446–5451. 14. Meisner J, Moran CPJ, Jr. (2011) A LytM domain dictates the localization of proteins to the mother cell-forespore interface during bacterial endospore formation. J Bacteriol 193:591–598. 15. Holm L, Kääriäinen S, Rosenström P, Schenkel A (2008) Searching protein structure databases with DaliLite v.3. Bioinformatics 24:2780–2781. 16. Firczuk M, Mucha A, Bochtler M (2005) Crystal structures of active LytM. J Mol Biol 354:578–590. 17. Janin J, Bahadur RP, Chakrabarti P (2008) Protein-protein interaction and quaternary structure. Q Rev Biophys 41:133–180. 18. Spreter T, et al. (2009) A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system. Nat Struct Mol Biol 16:468–476. 19. Yip CK, et al. (2005) Structural characterization of the molecular platform for type III secretion system assembly. Nature 435:702–707. 20. Morlot C, Uehara T, Marquis KA, Bernhardt TG, Rudner DZ (2010) A highly coordinated cell wall degradation machine governs spore morphogenesis in Bacillus subtilis. Genes Dev 24:411–422. 21. Pils B, Schultz J (2004) Inactive enzyme-homologues find new function in regulatory processes. J Mol Biol 340:399–404. 22. Goodenough DA, Paul DL (2009) Gap junctions. Cold Spring Harb Perspect Biol 1: a002576. 23. Maeda S, et al. (2009) Structure of the connexin 26 gap junction channel at 3.5 A resolution. Nature 458:597–602. 24. Cowtan K (2006) The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr 62:1002–1011. 25. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240–255.

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forespore. This is a redundant mechanism as neither protein is essential for engulfment in WT cells, but SpoIIQ and SpoIIIAH are required for engulfment in mutants in which the activity of the peptidoglycan degrading complex of SpoIID, SpoIIM, and SpoIIP (i.e., DMP) is reduced (11, 20). Finally, the SpoIIQ and SpoIIIAH interaction is required for σG activation upon completion of engulfment. The proposal that this involves the formation of an intercellular channel (1, 2), without precedent in bacteria, is lent further support by the simple modeling experiments carried out here. Bacterial LytM-type metalloproteases cleave the peptides that bridge glycan strands in the bacterial cell wall. The structural and sequence similarity of SpoIIQ to LytM and the localization of SpoIIQ to sites of peptidoglycan turnover suggested the possibility that SpoIIQ binds to and/or is regulated by cell wall peptides (14). There are many precedents for inactive enzyme homologues acquiring new functions as binding proteins (21). The crystal structure shows that the surface of SpoIIQ corresponding to the proposed substrate-binding groove in LytM is not occluded by the binding of SpoIIIAH (Fig. 3 and Fig. S2). This groove is, however, less prominent in SpoIIQ and closed off by the β2-β3 turn, which is involved in SpoIIIAH binding, and which covers the would-be catalytic center. The channel connecting the mother cell and the forespore during sporulation in B. subtilis may be compared with gap junction channels that facilitate intercellular transport and communication in multicellular organisms (22). The latter are formed by the head-tohead docking of hemichannels or connexons, consisting of connexin subunits in a hexameric ring. The structure of the connexin 26 gap junction channel has been determined by X-ray crystallography and EM, revealing that each connexin subunit has a core of four transmembrane helices (23). The structure of this homotypic connexin channel differs from the heterotypic channel formed by SpoIIQ and SpoIIIAH. The discrete extracellular domains of SpoIIQ and SpoIIIAH are more substantial than the extracellular elements in connexin 26, which consist of loops linking the transmembrane helices held together by disulphide bonds. The extracellular elements in connexin 26 extend 23 Å from the membrane surface and