Significance
The bacterial protein FtsZ polymerizes into protofilaments to create the cytokinetic ring responsible for directing cell division. Cellular levels of FtsZ are above the concentration required for Z-ring formation. Hence, FtsZ-binding proteins have evolved that control its spatiotemporal formation. The SlmA protein is one such factor that, when bound to specific chromosomal DNA, inhibits FtsZ polymerization to prevent Z rings from forming through the bacterial chromosome. This inhibition depends on complex formation between SlmA-DNA and the FtsZ C-terminal domain (CTD). Here we describe SlmA–DNA–FtsZ CTD structures. These structures and complementary biochemistry unveil the molecular basis for the unique requirement that SlmA be DNA-bound to interact with FtsZ, a mechanism that appears to be conserved among SlmA-containing bacteria.
Keywords: cell division, nucleoid occlusion, FtsZ, SlmA, protein–protein interaction
Abstract
Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called “nucleoid occlusion” (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator–CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA–DNA–FtsZ CTD regulatory interaction and provide insight into FtsZ–regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA–DNA–FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA–DNA–FtsZ interaction with implications for SlmA’s NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.
In Escherichia coli cell division is directed by a cytoskeletal element called the “Z ring,” which is formed at the cell membrane by the tubulin-like protein, FtsZ (1–5). FtsZ is an ancient and highly conserved protein that mediates cell division in most bacteria, many archaea, chloroplasts, and the mitochondria of primitive eukaryotes (5). FtsZ consists of three main domains: a globular core that harbors the GTP-binding site, a flexible C-terminal linker (CTL) that is ∼50 residues long in E. coli but is of variable length among FtsZ homologs, and a C-terminal domain (CTD) comprised of a highly conserved set of residues followed by a short and less conserved, variable (CTV) region (Fig. S1A) (6–9). FtsZ self-assembles into linear protofilaments in a GTP-dependent manner by interactions between its globular domains, and data suggest that loosely arranged lateral contacts between protofilaments mediate the formation of the Z ring at the cell center (10–16). Notably, the intracellular levels of FtsZ remain largely unchanged during the cell cycle and exceed the critical concentration required for Z-ring formation (17). A diverse repertoire of FtsZ-binding regulatory proteins has evolved that affect FtsZ localization and polymerization to ensure that the Z ring is created at the correct place and time during cell division (2–4, 18–20). In the model bacteria E. coli and Bacillus subtilis, two FtsZ regulatory systems, the Min system (21–25) and nucleoid occlusion (NO) (26–28), play key roles in controlling temporal and spatial establishment of the Z ring. The Min system prevents Z-ring formation at the cell poles, and NO prevents Z rings from forming over chromosomal DNA (Fig. S1B). Although the Min system has been studied extensively for decades, NO-associated factors have been discovered only recently, and the molecular mechanisms by which they function have remained unclear.
Fig. S1.
Schematic overview of FtsZ structure, function, and regulation. (A) Ribbon diagram of the FtsZ protofilament (PDB ID code 4DXD). One subunit is colored green (to aid in subunit delineation), and the other subunits are red. GTP molecules are not shown for clarity. FtsZ is composed of an N-terminal core region followed by a long CTL, which is attached to the CTD (shown as a box). (B) Mechanism of cell division site placement. Shown is a cartoon of a rod-shaped E. coli bacterial cell indicating the processes that direct Z-ring formation to the cell center. The cell poles are protected from Z-ring assembly by the Min inhibitory system. This system establishes a high local concentration of the FtsZ inhibitor MinC at the poles, thus preventing the generation of anucleate minicells. The replicated chromosomes are spared from Z-ring bisection by NO. The zone of NO is indicated by light blue regions surrounding the chromosomal DNA. SlmA binds to non-Ter chromosome regions to inhibit Z-ring formation.
In B. subtilis, a protein called Noc, which contains a putative ParB-like fold, participates in NO, whereas in E. coli the TetR family member SlmA has been shown to be involved in this process (29–38). Cellular studies on Noc and SlmA revealed that, although they belong to distinct structural families, they display similar NO properties in that both are synthetic lethal with a knockout of Min function and both localize to the nucleoid, where they inhibit Z-ring formation in their vicinity. Both SlmA and Noc also interact with specific DNA sequences, SlmA-binding sites (SBS) and Noc-binding sites (NBS), respectively. These DNA sites are located throughout the E. coli and B. subtilis nucleoids with the exception of the terminus-containing (Ter) region (31–33). The Ter is the last chromosomal region to segregate (39, 40). Hence, the exclusion of Noc and SlmA, which inhibit Z-ring formation, at the Ter region allows the Z ring to form at the cell center concomitant with DNA segregation (Fig. S1B). In this way, Noc and SlmA function as timing devices to coordinate DNA segregation with cell division. However, despite these similarities, the details of the NO mechanisms used by SlmA and Noc appear to be distinct. In particular, SlmA binds directly to FtsZ and antagonizes the formation of protofilaments. By contrast, Noc does not interact with FtsZ (29, 30, 33, 37, 38). Although data suggest that the FtsZ globular core may contribute to SlmA binding, the NO function of SlmA was shown to require its interaction with the CTD of FtsZ (37, 38). Hence, SlmA is a member of the expanding group of proteins that mediate their regulatory functions by binding the FtsZ CTD (37, 38). Proteins that interact with the FtsZ CTD are structurally and functionally diverse and in E. coli include ZipA, FtsA, ClpXP, MinC, and ZapD (2, 18–20). To date, only two structures of FtsZ regulatory protein–CTD complexes have been obtained: the E. coli FtsZ-binding domain of ZipA bound to the CTD and the Thermotoga maritima FtsA–CTD complex (41, 42). FtsA has an actin-like structure, whereas ZipA has a split βαβ fold, but the FtsZ CTD binds both proteins as a helix, suggesting that the FtsZ CTD may assume a helical state when binding to regulators. However, the CTDs of the E. coli and T. maritima FtsZ proteins are not well conserved, making it difficult to predict regulatory protein–CTD interactions based on these two structures alone. Therefore, how the FtsZ CTD can bind proteins that are structurally as well as functionally diverse remains unclear.
SlmA is unique among the characterized FtsZ CTD-binding regulatory proteins in that it must be bound to specific DNA to enable its interaction with the FtsZ CTD. Structures of SlmA showed that it harbors a canonical TetR-like fold (34, 43). Unlike other TetR proteins, SlmA functions not as a transcription regulator but rather as an NO factor. Previous studies showed that SlmA binds DNA as a dimer-of-dimers and can spread along the DNA (34). This finding was interesting in light of recent studies by the Lutkenhaus laboratory, which revealed that binding of SlmA requires FtsZ oligomerization because it converts the CTD into a multivalent ligand (38). The combined data, therefore, suggest that SlmA forms extended assemblages on the DNA, allowing it to bind simultaneously multiple FtsZ CTDs present on an FtsZ protofilament and ultimately antagonize protofilament formation. Although the SlmA–DNA–CTD interaction is central to the NO mechanism, the molecular basis for this interaction and, importantly, why SlmA must be bound to its SBS DNA sites to permit avid interactions with the FtsZ CTD are unknown. To gain insight into these questions and to further our understanding of FtsZ CTD interactions with regulatory proteins, we performed structural and biochemical analyses on SlmA–DNA–FtsZ CTD ternary complexes.
Results
Overall Structures of SlmA Complexes Bound to the FtsZ CTD.
To deduce the molecular basis for the requirement that SlmA must be bound to DNA to enable its interaction with the FtsZ CTD, we performed structural studies on SlmA–DNA–FtsZ CTD complexes from E. coli, Vibrio cholera, and Klebsiella pneumonia. The K. pneumonia and V. cholera SlmA proteins share 95% and 67% sequence identity with the E. coli protein, respectively, and their CTDs differ in only two residues: the E. coli and K. pneumonia CTD is DYLDIPAFLRKQAD, and that of V. cholera is GYLDIPAFLRRQAD. Previous analyses showed that these SlmA proteins all bind specifically to the SBS, 5′-GTGAGTACTCAC-3′ (34). To obtain complexes of SlmA–DNA with the CTD, SlmA–DNA solutions were mixed 1:1 (SlmA:CTD) with their respective FtsZ CTD 14-amino acid peptide (SI Materials and Methods), and crystallization trials were carried out. Structures of E. coli SlmA-12mer DNA, K. pneumonia SlmA-12mer DNA, and V. cholera SlmA-12mer DNA bound to the CTD were obtained at 2.6-, 3.0-, and 1.9-Å resolution, respectively (Fig. 1 and Table S1).
Fig. 1.
SlmA–DNA–FtsZ CTD ternary complexes. (A) Ribbon diagrams showing the overall structures of the SlmA–DNA–CTD complexes from V. cholera, E. coli, and K. pneumonia. SlmA dimers are colored cyan/yellow and orange/purple and the bound CTDs are shown as red stick surface renderings. (B) Fo-Fc electron density maps (white mesh) shown for each structure (labeled) calculated before the CTD had been added and contoured at 2.5 σ. (C, Left) Localization of the FtsZ CTD-binding site on SlmA. The FtsZ CTD is shown as red sticks bound between regions of the SlmA C domain, α4, α5, α6, and the DNA-binding domain, α1. (Right) An electrostatic surface representation of SlmA with bound peptide. Blue and red represent electropositive and electronegative regions, respectively. Shown is the V. cholera SlmA–DNA–FtsZ CTD, complex but all the structures harbor the same electrostatic surface properties.
Table S1.
Data collection and refinement statistics for SlmA–CTD and SlmA–DNA–CTD complexes
SlmA complex with CTD | EcSlmA-12-pep | KpSlmA-pep | VcSlmA-12-pep | KpSlmA-12-pep |
Space group | P212121 | P6522 | P3121 | P32 |
Cell dimensions | ||||
a, b, c, Å | 70.1, 158.9, 200.2 | 69.2, 69.2, 325.6 | 69.6, 69.6, 249.8 | 84.8, 84.8, 161.6 |
α, β, γ, ° | 90.0, 90.0, 90.0 | 90.0, 90.0, 120.0 | 90.0, 90.0, 120.0 | 90.0, 90.0, 120.0 |
Resolution, Å | 124.5–2.60 | 59.9–2.30 | 108.1–1.90 | 77.9–3.00 |
Rsym or Rmerge, % | 12.4 (44.3) | 6.7 (44.3) | 6.3 (37.6) | 12.7 (40.3) |
I/σI | 9.0 (2.8) | 11.2 (2.1) | 10.0 (1.9) | 8.0 (1.9) |
Completeness, % | 99.9 (99.5) | 100.0 (99.9) | 94.8 (73.4) | 94.1 (89.9) |
Redundancy | 5.8 (5.7) | 4.0 (4.0) | 3.3 (2.5) | 2.0 (1.9) |
Refinement | ||||
Resolution, Å | 124.5–2.60 | 59.9–2.30 | 108.1–1.90 | 77.9–3.00 |
Rwork/Rfree, % | 23.1/25.7 | 21.0/25.9 | 19.2/21.9 | 25.6/28.9 |
No. atoms | ||||
Protein | 13,620 | 3,341 | 3,712 | 6,937 |
Water | 215 | 105 | 238 | 23 |
rmsd | ||||
Bond lengths, Å | 0.002 | 0.003 | 0.006 | 0.009 |
Bond angles, ° | 0.572 | 0.699 | 0.955 | 1.340 |
Values in parentheses are for highest-resolution shell.
Each SlmA subunit in the SlmA–DNA–CTD ternary structures is composed of an N-terminal DNA-binding domain, with a helix-turn-helix motif formed by helices 2 and 3, and a C-terminal dimer domain that is comprised of six helices, α4–α9 (32, 34). The SlmA molecules in the ternary complexes bind the SBS as a dimer-of-dimers. A comparison of the SlmA dimer-of-dimers in the ternary structures with previous SlmA–DNA complexes revealed no significant structural differences; superimpositions of corresponding Cα atoms resulted in rmsds of 0.6–0.8 Å (Fig. S2A). The SlmA–DNA interaction is allosteric, whereby binding of the first SlmA dimer induces significant DNA distortion that enhances binding of the second dimer (34). The DNA distortion is a sharp kink mediated by the insertion of the conserved residue Thr33 (Thr31 in V. cholera SlmA) between phosphate groups of the DNA backbone. This DNA deformation permits the specific pattern of contacts between SlmA and the DNA. These same contacts are observed in the SlmA–DNA–CTD ternary complexes (Fig. S2B). Thus, these results indicate that the binding of the FtsZ CTD to SlmA–DNA does not cause any significant structural changes in the SlmA subunits or in the overall SlmA–DNA complexes. Although SlmA proteins from E. coli, K. pneumonia, and V. cholera were used in these studies, and distinct crystal forms were obtained, clear density for the FtsZ CTD was observed in the same location in all the structures (Fig. 1B). Notably, this extended, surface-exposed CTD-binding site is distinct from the compact and largely buried pocket that all other characterized TetR proteins have been shown to use for ligand binding (Fig. 2A). Ligand binding to the small pocket in TetR transcription regulators induces structural changes that deactivate their DNA-binding functions, allowing them to act as metabolic sensors (43). By contrast, SlmA must be bound to the DNA to interact with its ligand, the FtsZ CTD, and data show that it remains bound to the DNA subsequent to CTD binding. Indeed, its NO function demands that it not be induced from the DNA. Hence, the lack of conformational changes observed in the structures upon CTD binding is consistent with the NO function of SlmA.
Fig. S2.
Comparison of SlmA–DNA and SlmA–DNA–FtsZ CTD ternary complexes. (A) SlmA–DNA–CTD complexes (yellow) from E. coli, K. pneumonia, and V. cholera are superimposed onto structures solved in the absence of CTD (CTD-free) (34). The CTDs are shown as yellow surfaces. The superimpositions resulted in rmsds of 0.6–0.8 Å for corresponding Cα atoms, indicating that FtsZ CTD binding causes no structural changes to the binary structures. (B) Close-up view of the DNA-binding residues and their DNA contacts in the CTD-bound and free forms (colored as in A). Again, no significant structural changes are induced in DNA binding by CTD interaction with SlmA–DNA.
Fig. 2.
The FtsZ CTD binds to a surface-exposed pocket in the SlmA DNA-bound state. (A) SlmA binds its CTD ligand in a pocket distinct from other TetR proteins. (Left) V. cholera SlmA–CTD structure showing the location of one bound CTD (red space-filling). The surface-exposed region that binds the CTD is colored yellow. (Right) Structure of a typical TetR protein, QacR, with the ligand bound in the mostly buried C-domain pocket. Ligand-binding residues are colored yellow. (B) Superimposition of the C-terminal dimer domain of apo SlmA structures (blue, red, and green) onto the DNA-bound conformation (yellow) underscoring the flexibility of the apo form of SlmA. The CTD is shown as a yellow dot surface. (C) Overlay of SlmA–DNA–CTD complexes from multiple crystal forms and species (E. coli, K. pneumonia, and V. cholera) showing the conservation of the SlmA structure and the conformation of the bound FtsZ CTD. Notably, the K. pneumonia SlmA–CTD complex (green) that was solved in the absence of DNA adopts the same DNA-bound state.
In the V. cholera and K. pneumonia structures all the SlmA subunits are complexed to the CTD (Fig. 1A). However, only one subunit in the E. coli SlmA structure is bound to the FtsZ CTD peptide, likely because of the high salt conditions required for crystallization. This finding is notable, however, because it indicates that CTD binding to SlmA is not cooperative. Strikingly, the CTD does not bind the SlmA–DNA complex as a helix, as has been observed in other FtsZ regulatory protein–CTD complexes, but adopts a fully extended conformation whereby it binds along nearly the entire length of a SlmA subunit. The acquisition of this elongated, loop-like structure allows the CTD to insert deeply into a surface cavity on each SlmA subunit, which is quite narrow (Fig. 1). The extended state of the peptide also permits multiple contacts to both the SlmA CTD and DNA-binding domain; the CTD N-terminal region interacts with residues from α4, α5, and α6 on the SlmA C-terminal dimer domain (referred to herein as either the “C domain” or “dimer domain”), whereas the C-terminal residues of the CTD interact with amino acids from α1 on the SlmA DNA-binding domain.
Basis for the DNA Requirement for CTD Binding to SlmA.
Studies have demonstrated that specific DNA binding by SlmA is required for efficient interaction with the FtsZ CTD (36, 38). Multiple structures of apo SlmA have been obtained from V. cholera, K. pneumonia, and E. coli and have captured conformations in which the DNA-binding domains exist in a range of orientations (Fig. 2B) (34). In some of the structures the DNA-binding domains are significantly disordered, but in others a conformation very similar to the DNA-bound conformation was trapped in the crystal. It is thought this flexibility in the apo form, as in other TetR proteins, allows SlmA to adjust optimally and dock on the DNA (43). Therefore, apo SlmA does not adopt a specific structural state; instead, the DNA-binding domains are dynamic when SlmA is not complexed to specific DNA (Fig. 2B). DNA binding locks in a specific conformation, as revealed by the finding that all SlmA structures have an essentially identical conformation in multiple SlmA–DNA structures (34). The SlmA–DNA–CTD ternary complex structures explain why DNA complexation is crucial to permit FtsZ CTD binding, because it shows that the specific DNA-bound state of SlmA harbors the correct juxtaposition of α1, from the DNA-binding domain, with α4–α6 from the dimer domain needed for proper CTD docking. Support for this notion comes from a structure of K. pneumonia SlmA–CTD obtained in the absence of DNA. Although DNA was not present in this structure, clear density was revealed for the CTD bound in the identical location as in the SlmA–DNA–CTD complexes in a subunit that had clearly adopted the DNA-bound state (Figs. 1 A and B and 2C).
The FtsZ CTD Binds SlmA in an Extended Conformation.
The FtsZ CTD-binding site on SlmA formed at the junction between the DNA-binding site and the C domain is surprisingly hydrophobic in character. It also is quite narrow and hence could not be accessed by a helix. However, the extended conformation that is adopted by the SlmA-bound CTD allows hydrophobic residues exposed on the CTD surface to fit perfectly into the elongated SlmA cavity and evade solvent. Specifically, the CTD peptide makes five hydrophobic contacts from residues (using E. coli numbering) Ile374, Pro375, Phe377, and Leu378 to residues in the SlmA hydrophobic crevice (Fig. 3A). FtsZ CTD residue Phe377 (V. cholera residue Phe392) appears to play a central role in binding to the specific DNA-bound state of SlmA, because it inserts in a hydrophobic cavity formed at the interface between the DNA-binding domain and the C domain that is optimally shaped to fit the phenyl side chain (Fig. 3A). Within this cleft, the FtsZ CTD Phe377 side chain interacts with Leu16 and Ala20 and the aliphatic atoms of Gln17 from α1 in the DNA-binding domain, whereas its other face is encased by SlmA C-domain residues Leu61, Phe65, and Leu105 (Fig. 3A). In addition to Phe377, Leu378 also contacts residues within the SlmA DNA-binding domain, including Ala20 and the aliphatic atoms of residues Glu21 and Glu24. The remainder of the bound FtsZ CTD residues interacts with the SlmA C domain. These interactions include contacts between the CTD residue Pro375 and SlmA residue Phe65 and the aliphatic portions of the Ser69 and the Phe98 side chains. SlmA residue Arg73 is anchored over Pro375, thus shielding it from solvent, via a salt bridge with CTD residue Asp373. Finally, CTD residue Ile374 embeds into the narrow hydrophobic SlmA cavity and contacts Phe98 and Leu94.
Fig. 3.
(A) Close-up view of FtsZ CTD interactions with E. coli and K. pneumonia SlmA (Left), which are identical, and V. cholera SlmA (Right). Residues shown via genetics to be required for the inhibitory effect of SlmA on the formation of FtsZ protofilaments (35) are colored cyan and indicated by asterisks. (B) Sequence alignment of the SlmA proteins used in structural studies highlighting the residues involved in FtsZ binding. Residues highlighted in cyan, green, and gray correspond to those making hydrophobic, salt bridges, and hydrogen bonds, respectively, to CTD residues. Identical residues are indicated by asterisks.
In addition to hydrophobic contacts, there are several hydrogen bonds and salt bridges in the SlmA–CTD complex. As noted, CTD residue Asp373 makes a salt bridge to SlmA residue Arg73. Residue Asn102, from the SlmA C domain, hydrogen bonds to amide nitrogen and carbonyl oxygen atoms of CTD residues Ala376 and Phe377. Similarly, SlmA residue Gln17, from the DNA-binding domain, contacts the backbone atoms of FtsZ CTD residues Phe377 and Arg379. These backbone contacts are key, because they specify the extended nature of the peptide and anchor it into the pocket. The CTD Arg379 residue is also juxtaposed with the highly acidic side of α1 in the DNA-binding domain (Fig. 1C). In the E. coli, V. cholera, and K. pneumonia SlmA–DNA–CTD complexes there is no density for residues C-terminal to CTD residue Arg379, and only weak density is observed in the K. pneumonia SlmA–CTD complex for Arg379 and Lys380. This finding indicates that SlmA proteins do not interact with the CTV region and bind CTD residues 370–380, which correspond to the conserved N-terminal region of the CTD, and suggests that SlmA proteins are active in bacteria that contain FtsZ proteins with CTVs that are highly variable in length and sequence. Moreover, it shows that SlmA shows binding selectivity for a relatively short sequence, LDIPAFL. BLAST searches indicate that this sequence is not found in other E. coli proteins, ensuring that the SlmA–CTD interaction is unique.
Conserved Model for SlmA–FtsZ CTD Interaction in Bacterial NO.
Residues that contact the FtsZ CTD are conserved among E. coli, K. pneumonia, and V. cholera SlmA proteins. V. cholera SlmA contains an alanine (Ala95) in the place of E. coli/K. pneumonia residue Gly97 and a glutamate, Glu19, instead of Leu21. These substitutions do not affect CTD binding, because the alanine side chain in V. cholera protein is small enough to permit docking of the CTD, and the Glu19/Leu21 side chain mediates contacts with the CTD via side-chain Cβ atoms. Further, although the V. cholera FtsZ CTD differs from the E. coli and K. pneumonia FtsZ CTDs at residues 370 and 380 (the residues are glycine and arginine, respectively, in V. cholera and are aspartic acid and lysine, respectively, in E. coli/K. pneumonia), the structures show that these residues are not involved in SlmA binding. SlmA homologs appear to be widespread and are present in γ- and β-proteobacteria with the exceptions of Francisella, Legionella, Pseudomonas, Stenotrophomonas, Xanthomonas, and Neisseria. Multiple sequence alignments of SlmA proteins (ranging from 98% to 48% identity) show that the key residues involved in CTD binding are conserved in the SlmA proteins and that residues that make important contacts are either conserved or contain conservative substitutions (Fig. S3). Sequence alignments of the corresponding FtsZ CTDs show that SlmA-contacting residues are identical. Thus, these data indicate that the SlmA–FtsZ CTD interaction, and hence its NO function, are likely to be conserved across SlmA-containing bacteria.
Fig. S3.
Multiple sequence alignment of SlmA proteins and their corresponding FtsZ CTD. (Upper) Sequence alignment of multiple SlmA proteins. Helix-turn-helix (HTH) DNA-binding residues are highlighted in cyan (34); residues involved in FtsZ CTD binding are highlighted in yellow; CTD-interacting residues that the structure indicates are particularly critical for binding are boxed. Secondary structural elements revealed in the SlmA structures are shown above the sequences. Asterisks below the alignments indicate identical residues. (Lower) Sequences of the FtsZ CTD of the organisms in the Top, Upper, panel, shown in the same order. Note: the FtsZ CTD region that is bound by SlmA (boxed) is 100% conserved in these FtsZ proteins. The bacterial species identity follows the code: gi|254772829, E. coli; gi|254772839, Erwinia tasmaniensis; gi|73621891, Photorhabdus luminescens subsp. laumondii TTO1; gi|166977644, Serratia proteamaculans 568; gi|166977657, Yersinia enterocolitica subsp. enterocolitica 8081; gi|73621903, Yersinia pestis; gi|254772842, Proteus mirabilis HI4320; gi|73621892, Photobacterium profundum; gi|166977655, Vibrio campbellii ATCC BAA-1116; gi|73621901, Vibrio vulnificus CMCP6; gi|166977651, Shewanella sp. ANA-3; gi|166977650, Shewanella putrefaciens CN-32; gi|122298389, Shewanella frigidimarina NCIMB 400; gi|254772853, Shewanella piezotolerans WP3; gi|123166544, Shewanella denitrificans OS217; gi|189046725, Shewanella sediminis HAW-EB3; gi|166977649, Shewanella loihica PV-4; gi|357580492, Marinomonas sp. MWYL1; gi|357580491, Marinobacter hydrocarbonoclasticus VT8.
Probing the SlmA–DNA–CTD Structure.
Several studies have probed the SlmA–FtsZ CTD interaction and its effect on FtsZ filament formation (35, 37, 38). A genetic screen carried out by Cho et al. (35) revealed that residues Phe65, Arg73, Leu94, Gly97, Arg101, and Asn102 were critical for the antagonism of FtsZ protofilament formation by E. coli SlmA. These residues map precisely to the CTD portion of the FtsZ CTD-binding pocket revealed in our structures (Fig. 3A). Interestingly, this study did not identify residues in α1, likely because the side-chain aliphatic portion of α1 residues mediates most of the contacts to the CTD. In a separate study, FtsZ mutations L378E and I374K were shown to abrogate the SlmA–FtsZ interaction (37). The SlmA–DNA–CTD structures show that these substitutions would result in the insertion of large and charged residues into the hydrophobic CTD-binding crevice on SlmA; such an insertion would prohibit binding. Furthermore, our structures show that SlmA interacts with the same residues as ZipA, as is consistent with studies showing that ZipA competes with SlmA for FtsZ CTD binding (38). However, to probe the model further, we sought to test two specific structure-based predictions: that Phe377 is critical for the DNA-specific SlmA–CTD interaction and that the FtsZ CTV, which is not visible in the structure and does not contribute to binding, would be dispensable for the interaction.
Studies showed that the full-length FtsZ protein, which exposes multiple CTDs as a multivalent ligand source, binds its regulators with significantly enhanced affinity (38). Indeed, the FtsZ CTD must be present as a multivalent ligand to observe appreciable binding at the relatively low concentrations used in biochemical measurements (38). Thus, we developed an assay that used a chimeric construct in which the FtsZ 50-residue linker–CTD region was connected to C terminus of the tetramerization domain of human p53 to probe our structural model (44). This construct and mutant forms of the construct then were titrated into preformed complexes of SlmA bound to fluorescently labeled DNA. Binding resulted in a saturable increase in fluorescence polarization (FP) that could be used to determine an apparent Kd. Following this strategy, a p53 chimera containing the wild-type FtsZ CTD sequence bound the E. coli SlmA–DNA complex with an apparent Kd of 25.6 ± 1.8 μM (Fig. 4A). When the experiment was performed with a chimera lacking the CTD (ΔCTD), i.e., with the p53 tetramerization domain alone, no binding was observed. Removal of the CTV (ΔCTV) resulted in binding similar to that observed for the wild-type chimera (apparent Kd = 27.0 ± 1.4 μM), whereas a single F377A mutation in the CTD resulted in only weak, nonsaturable binding (Fig. 4A). Thus, the combined results from the FP binding studies support the SlmA–DNA–CTD structural models. The wild-type SlmA–DNA–CTD binding affinity obtained by this assay is comparable to those previously measured for FtsZ CTD binding to other regulatory proteins via biosensor assays, which present multiple proteins on a surface (41, 42). For example, ZipA and FtsA were shown to bind the FtsZ CTD with Kds of 20 μM and 50 μM, respectively (41, 42).
Fig. 4.
SlmA–DNA–CTD complex: CTD conformational adaptability and insight into SlmA-mediated NO. (A) FP isotherms of SlmA–DNA binding to various p53 tetramer domain–FtsZ linker–FtsZ CTD chimeras to test the SlmA–DNA–FtsZ CTD structural model. (B) Comparison of FtsZ CTD conformation in structures solved bound to FtsZ regulators. (Upper) Schematic of the FtsZ domain organization. Residues D373 (D338 in T. maritima) are aligned for reference and underscore the dramatic range of conformational states adopted by the CTD. (Lower) Sequence alignment of the FtsZ CTDs from E. coli, K. pneumonia, V. cholera (analyzed in this study), and T. maritima. CTT, conserved region of the CTD. Residues conserved in all CTDs are colored blue, and those conserved in the E. coli, K. pneumonia, and V. cholera CTDs are colored blue-green. Note: Only the conserved residues make contact with SlmA (Fig. 3A). (C) SlmA-mediated NO. This process involves SlmA binding to the SBS as a dimer-of-dimers and nucleating the binding of adjacent SlmA dimers (34). Then FtsZ filaments are recruited to the nucleoid by binding SlmA via their CTD (shown as loops) and subsequently FtsZ core regions (squares). DNA charge might contribute to protofilament fracturing. Once the protofilaments are disrupted into subunits, they no longer bind SlmA–DNA with significant affinity and diffuse from the nucleoid. (D) Schematic of E. coli cell division with Min- and NO-mediated NO processes indicated. SlmA binding to the nucleoid non-Ter regions averts nucleoid fragmentation by FtsZ, whereas the Min system prevents Z-ring formation at the poles, the net effect being to drive Z-ring formation at the cell center.
The FtsZ CTD: An Intrinsically Disordered Region Capable of Adopting Multiple Conformations upon Binding Diverse FtsZ Regulatory Proteins.
The FtsZ CTD mediates the majority of its contacts with regulatory proteins. As a result, it has been called the FtsZ “landing pad” (18). How these proteins with diverse structures and functions can bind the same FtsZ CTD region has been a fascinating but unresolved question. Currently the only proteins whose structures have been obtained in the presence of the CTD are E. coli ZipA and T. maritima FtsA, both of which function to recruit FtsZ to the membrane (41, 42, 45). The CTD binds both these proteins as helices. In the ZipA–CTD structure most of the contacts made by the CTD are hydrophobic. In contrast, in the FtsA–CTD complex there are only three contacts, and all are salt bridges (41, 42). It is possible that the differences in these complexes might reflect the divergence in the FtsZ CTD between these two organisms; the T. maritima and E. coli CTD share only five residues in common, and the T. maritima CTD harbors a longer CTV: the T. maritima CTD is PEGDIPAIYRYGLEGLL compared with the E. coli FtsZ CTD, DYLDIPAFLRKQAD (conserved residues are underlined). Indeed, although the helical segment of the CTD bound to both E. coli ZipA and T. maritima FtsA begins with the conserved proline (Pro375 in E. coli numbering), the helix bound to FtsA has a kink caused by a glycine that is not present in the E. coli CTD (Fig. 4B). Hence, the question of how the FtsZ CTD can bind so many diverse regulatory proteins has remained unanswered.
Our structures of SlmA–DNA bound to the FtsZ CTD resolve this issue. Importantly, these structures show that the CTD does not bind all its regulators as a helix but can adopt a striking range of conformations depending on its binding partner (Fig. S4). Indeed, although the CTD contains a significant number of hydrophobic residues, SlmA and ZipA use distinct strategies to shield these residues from solvent. The hydrophobic face of the amphipathic CTD helix bound to ZipA is docked into a large hydrophobic surface on ZipA, whereas in SlmA an extended, narrow cavity with hydrophobic character allows the insertion of the hydrophobic CTD residues. As noted, despite the differences in the conformation by which the CTD binds these regulators, the affinities of the CTD for these proteins are similar (41, 42). As is consistent with this affinity, analysis of the buried surface areas showed that the ZipA–CTD and FtsA–CTD complexes bury 565 and 570 Å2, respectively, whereas the SlmA–CTD interaction buries 633–727 Å2. However, these combined findings suggest the unlikelihood of predicting how the FtsZ CTD might bind each of its regulators, even if biochemical information, such as binding affinities, is available, and indicate that structures likely will be required to deduce the binding mechanisms for each complex.
Fig. S4.
FtsZ CTD binds structurally diverse regulatory proteins by adopting multiple conformations. Comparison of the structures that have been solved of the FtsZ CTD bound to regulatory proteins. The proteins are colored cyan, and the FtsZ CTD is colored purple. Notably, the structures of the proteins are diverse, and the FtsZ CTD binds each by adopting strikingly different conformations.
SI Materials and Methods
Protein Expression and Purification.
The genes encoding the E. coli, K. pneumonia, and V. cholera SlmA proteins were purchased from GenScript Corporation (www.genscript.com) and subcloned into pET15b such that His-tags were expressed for purification. For protein expression, the slmA encoding pET15b vectors were transformed into C41(DE3) cells. All SlmA constructs (E. coli, K. pneumonia, and V. cholera) were expressed and purified using the same procedure. Briefly, overnight cultures grown with an slmA expression pET15b vector were used to inoculate 9 L of Luria broth (LB) with 100 μg/mL ampicillin. Inoculated cells were grown to an OD600 = 0.5 at 37 °C and induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h. The cells then were resuspended in 20 mM Tris (pH 7.5), 300 mM NaCl, 10% glycerol, 1 mM β-mercaptoethanol, 2 mg/L DNase I, and 1 mM PMSF and were lysed with a microfluidizer. Cell debris was removed by centrifugation for 1 h at 4 °C and 34,960 × g. The SlmA-containing supernatant was adsorbed onto an Ni-NTA column and washed with buffer A [20 mM Tris (pH 7.5), 300 mM NaCl, 10% glycerol, and 1 mM β-mercaptoethanol] and 10 mM imidazole. SlmA was eluted using buffer A containing 0.25–1 M imidazole. All SlmA proteins were >90% pure after the Ni-NTA step. The His-tags were removed via thrombin cleavage. Then the protein was buffer exchanged using a Centricon 30-kDa centrifugal filter concentrator, resulting in the removal of the excised tag.
Crystallization and Structure Determination of the E. coli SlmA–CTD Complex.
To form the E. coli SlmA–DNA–FtsZ CTD complex, E. coli SlmA (at 20 mg/mL) was mixed at a ratio of two SlmA dimers to one 12mer SBS-containing DNA duplex, 5′-GTGAGTACTCAC-3′, based on the binding stoichiometry determined from previous studies. FtsZ peptide (DYLDIPAFLRKQAD) was added to this mixture at a final concentration of 2 mM, and crystals were grown by combining this solution 1:1 with a crystallization reagent consisting of 2.5 M NaCl, 0.1 M Hepes (pH 7.5). The crystals grew in 1–3 d, take the space group P212121 with a = 70.1 Å, b = 158.9 Å, and c = 200.2 Å, and diffracted to 2.6-Å resolution. The crystals were cryopreserved in a solution consisting of the crystallization reagent supplemented with 20% glycerol. X-ray intensity data were collected at Advanced Light Source (ALS) beamline 8.3.1 and were processed with MOSFLM. For structure determination, an SlmA dimer-of-dimer-DNA complex (PDB ID code 4GCL) was used in molecular replacement with the program MolRep after truncating the 14mer DNA to 12 bp. Two solutions were obtained, showing that there are two SlmA dimer-of-dimer–DNA complexes in the crystallographic asymmetric unit (ASU). Rigid body refinement followed by xyzb refinement was performed, after which clear density for the FtsZ CTD, specifically residues 372–382, was observed in one subunit. Electron density was observed near the CTD-binding pocket in some of the other subunits but was weak and hence was not included. The structure was refined using Phenix. Ramachandran analyses showed that 97.2% of the residues are in the most favored region with no outliers (47). See Table S1 for final crystallographic statistics.
Crystallization and Structure Determination of K. pneumonia SlmA–DNA–FtsZ CTD and SlmA–CTD complexes.
To form K. pneumonia SlmA–DNA–FtsZ CTD complexes, K. pneumonia SlmA (at 30 mg/mL) was mixed as for the E. coli complex with 12mer SBS DNA, 5′-GTGAGTACTCAC-3′, and FtsZ peptide was added to a final concentration of 2.5 mM. Crystals were grown by mixing this SlmA–DNA–FtsZ CTD solution 1:1 with a crystallization reagent consisting of 29% PEG 3000, 0.1 M Tris HCl (pH 7.0), 0.2 M NaCl. Crystals took 2–3 wk to grow and take the space group P32 with a = b = 84.8 Å and c = 161.6 Å. The crystals were cryopreserved straight from the drop. Intensity data were collected to 2.8-Å resolution at ALS beamline 8.3.1 and were processed with MOSFLM. For structure determination, the K. pneumonia dimer-of-dimer–DNA complex (PDB ID code 4GCK) was used for molecular replacement in MolRep. The ASU contains one SlmA dimer-of-dimer–DNA complex. After initial refinement (rigid body followed by positional/thermal parameter refinement), clear density for FtsZ CTD peptide residues 372–379 was evident in all the SlmA subunits in the ASU. After inclusion of the CTD residues, final refinement was performed. See Table S1 for final refinement statistics.
Crystals of an apo K. pneumonia SlmA–CTD complex were also obtained by adding the FtsZ CTD to a final concentration of 3 mM to a solution of 40 mg/mL K. pneumonia SlmA and mixing this complex 1:1 with a solution of 600 mM Na phosphate, 1,200 mM potassium phosphate, 0.1 M imidazole (pH 8.0), 0.2 M NaCl. The crystals take the hexagonal space group P6522 with a = b = 69.2 Å and c = 325.6 Å and were cryopreserved from the drop. Data were collected at ALS beamline 8.3.1 and were processed with MOSFLM. Molecular replacement (using the K. pneumonia SlmA structure, PDB ID code 4GFL, as a search model) revealed that the ASU is comprised of one SlmA dimer. Although DNA was not present in this structure, clear density for FtsZ CTD residues 372–380 was observed in one SlmA subunit, which had adopted the DNA-bound conformation. The structure was refined using Phenix (47). Ramachandran analysis showed that 97.2% of the residues are in the most favored region of the Ramachandran plot, and there were no outliers. See Table S1 for final crystallographic statistics.
Crystallization and Structure Determination of the V. cholera SlmA–DNA–FtsZ CTD Complex.
V. cholera SlmA–DNA–CTD mixtures were formed as described for the E. coli and K. pneumonia complexes, using a ratio of two SlmA dimers to one duplex DNA and adding 3 mM V. cholera CTD peptide. Crystals were grown by mixing this solution 1:1 with 10% PEG 8000, 0.1 M imidazole (pH 8.0), 0.1 M CaCl2. Crystals were cryopreserved straight from the drop. Data were collected to 1.9-Å resolution at ALS beamline 8.3.1 and were processed with MOSFLM. The crystals take the space group P3121 with a = b = 69.6 Å and c = 249.8 Å. For structure determination, the V. cholera SlmA–DNA structure (PDB ID code 4GCT) was used in molecular replacement after pruning the DNA to 12 bp. The Matthew's coefficient (VM) of the crystal suggested that an SlmA dimer and DNA half-site was the likely ASU. This supposition was confirmed in MolRep, which produced a clear solution; crystallographic symmetry generates the SlmA dimer-of-dimer–DNA complex. After initial refinement, clear electron density was evident for CTD residues 372–379 in one subunit and residues 370–379 in the other SlmA subunit. The model displays excellent stereochemistry with 99.2% of the residues in the most favored region of the Ramachandran plot and no outliers (47) (Table S1).
Generation and Purification of p53 Tetramerization Domain–FtsZ linker–CTD–CTV constructs.
Because detectable binding of SlmA–DNA to the CTD in biochemical assays requires that the CTD be present as a multivalent ligand, we generated a chimera that encodes the human p53 tetramerization domain (44) at the N terminus followed by the E. coli FtsZ C-terminal region encompassing the 50-residue FtsZ linker, followed by the CTD, to use in experiments to assess the effects of CTD mutations on SlmA–DNA binding. The chimera was subcloned into the pET15b vector for expression. This chimera and chimeras containing mutations in the CTD were all expressed to high levels and were soluble. To express the proteins, overnight cultures grown with chimeric expression pET15b vectors were used to inoculate 9 L of LB with 100 μg/mL ampicillin. The cells were grown to an OD600 = 0.5–0.7 at 37 °C and were induced with 0.5 mM IPTG at 37 °C for 4 h. The cells were resuspended in 20 mM Tris (pH 7.5), 300 mM NaCl, 10% glycerol, 1 mM β-mercaptoethanol, 2 mg/L DNase I, and 1 mM PMSF and were lysed using a microfluidizer. Cell debris was removed by centrifugation. The chimera-containing supernatant was applied to a Ni-NTA column and washed with buffer A and 10 mM imidazole. Proteins were eluted using buffer A containing 0.25–1 M imidazole and were >97% pure after this step. Size-exclusion chromatography and crosslinking with glutaraldehyde showed that the constructs formed higher-order oligomers (consistent with tetramers). The proteins were concentrated to 30 mg/mL for use in FP studies.
FP-Binding Assays.
FP assays were performed using a PanVera Beacon FP system. FP assays were carried out in a binding buffer consisting of 150 mM NaCl and 25 mM Tris⋅HCl (pH 7.5). E. coli, K. pneumonia, and V. cholera SlmA proteins were assessed for activity by FP assays by titrating increasing concentrations of SlmA into binding buffer containing 1 nM fluoresceinated SBS DNA (5′-GTGAGTACTCAC-3′). These assays revealed that the proteins retained activity for >1 mo. SlmA proteins all bind the DNA with Kds of ∼100 nM as previously reported (34). To assess the effects of FtsZ CTD amino acid substitutions on SlmA–DNA binding, p53 tetramerization domain–FtsZ(linker–CTD) constructs were produced. In these experiments, SlmA was first titrated into the binding buffer with 1 nM fluoresceinated DNA until saturation was reached. Then increasing concentrations of the p53 tetramerization domain–FtsZ(linker–CTD) were added. Binding was analyzed for a construct without the CTD, a construct containing a wild-type CTD, a construct containing a single F377A mutation in the CTD, and a construct missing CTD residues 381–383. The resultant binding isotherms were plotted and fit using KaleidaGraph.
Discussion
The FtsZ protein mediates cell division in most prokaryotes and is subject to regulation at multiple levels to ensure that the Z ring forms at the correct time and place during cell division. NO, the phenomenon by which the chromosomal DNA is spared from Z-ring bisection, is particularly important because Z-ring formation through the nucleoid would result in chromosome fragmentation. Thus, it is not surprising that failsafe mechanisms, including dedicated NO factors, have evolved to prevent this catastrophe. SlmA was identified as the E. coli NO factor (30) and was shown to inhibit FtsZ directly from forming Z rings through the DNA. This inhibition depends on an interaction between SlmA–DNA and the CTD of FtsZ (33, 37). Because FtsZ must be tethered to the membrane by interactions between its CTD and either ZipA or FtsA to form a Z ring, the SlmA–CTD interaction might prevent Z-ring formation simply by competition. However, studies showed that SlmA–DNA complexes actively disrupt FtsZ protofilaments, suggesting a more complex mechanism (33, 37). Data indicate that the FtsZ core also interacts weakly with SlmA–DNA subsequent to CTD binding. Interestingly, small-angle X-ray–scattering studies suggested that the FtsZ core also may bind the SlmA C domain; however, the resolution of the analyses was too low to assign a precise binding location (34). These interactions could collaborate to antagonize the formation of FtsZ protofilaments. SlmA can spread from its initial DNA site to form extended assemblages on the DNA. Thus, the SlmA assemblies could interact with multiple FtsZ molecules on a protofilament to fragment and antagonize its formation (Fig. 4 C and D). Once the protofilaments have been disrupted into single or small protofilaments, they would no longer bind SlmA–DNA, because this binding requires a multivalent form of the FtsZ CTD found only on the protofilaments (Fig. 4C). The requirement that SlmA be bound to the nucleoid to be active for this recruitment step is vital, because it prevents any free SlmA, from binding FtsZ protofilaments and disrupting Z-ring formation. The multiple SlmA–FtsZ interactions would bring FtsZ protofilaments within proximity of the electronegative nucleoid DNA (Fig. 4C). Hence, it is possible that the nucleoid could be involved in NO (Fig. 4 C and D). Indeed, charge has been shown to play a key role in the formation and clustering of FtsZ protofilaments. For example, the electropositive B. subtilis FtsZ CTD participates in bundling protofilaments (46), and although the E. coli FtsZ CTD does not appear to play a role in protofilament bundling, multiple studies have shown that cations and positively charged molecules enhance its polymerization (5).
Although more studies are needed to assess the specific roles played by SlmA and the nucleoid in the complex process of NO, a clearly vital step in SlmA-mediated NO is the recruitment of FtsZ and its tethering to the nucleoid by the SlmA–DNA–FtsZ CTD interaction. However, the molecular basis behind this interaction has been unknown. This interaction also is of interest because it is unusual among FtsZ regulators, in that it requires SlmA to be specifically bound to DNA. Here we deciphered the molecular mechanism behind this unique regulatory function by determining crystal structures of multiple SlmA–DNA–FtsZ CTD ternary complexes. Notably, all the structures, obtained under diverse crystallization conditions and with SlmA proteins from different organisms, revealed the same binding mode in which the FtsZ CTD interacts with the DNA-bound form of SlmA as an extended loop-like structure. This mode of ligand interaction is completely different from that used by other characterized TetR proteins to bind ligands, namely via a small, mostly buried cleft located in their C domains (43). Importantly, the SlmA–DNA–CTD structures also reveal that SlmA must be in its DNA-bound form to bind the CTD because it is locked into a particular conformation that harbors the correct juxtaposition of the dimer and DNA-binding domains that allows specific docking of the FtsZ CTD. Residues critical for CTD interactions are shared among SlmA proteins, indicating that they use a conserved mode of CTD binding. Finally, these findings indicate that the FtsZ CTD is able to bind a diverse array of regulatory proteins because it is able to adopt different conformational states depending on its regulatory binding partner. Thus, our structures not only reveal the underlying molecular mechanism by which SlmA–DNA recruits FtsZ to the nucleoid but also unveil how the FtsZ CTD acts as a landing pad for diverse regulatory proteins.
Materials and Methods
SI Materials and Methods provides details and additional methods not provided below.
The genes encoding the E. coli, K. pneumonia, and V. cholera SlmA proteins were purchased from GenScript Corporation and subcloned into pET15b so that His-tags were expressed for purification. For details of purification, crystallization, structure determination, and refinement (47), see SI Materials and Methods.
Acknowledgments
This work was supported by NIH Grant GM115563 (to M.A.S.). Beamline 8.3.1 at the Advanced Light Source is operated by the University of California Office of the President Multicampus Research Programs and Initiatives Grant MR-15-328599 and by the Program for Breakthrough Biomedical Research, which is partially funded by the Sandler Foundation.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: Crystallography, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 5HSZ, 5HBU, and 5HAW).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1602327113/-/DCSupplemental.
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