ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments

A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0) pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.     

ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains

FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division in Escherichia coli. FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro. In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring. ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ. Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo. The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA. Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro. Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein. Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA(-) cells. Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function. Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.     

The Escherichia coli cell division protein ZipA forms homodimers prior to association with FtsZ

ZipA is an essential component of the cell division machinery in E. coli and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.     

ZipA is a MAP-Tau homolog and is essential for structural integrity of the cytokinetic FtsZ ring during bacterial cell division

The first visible event in prokaryotic cell division is the assembly of the soluble, tubulin-like FtsZ GTPase into a membrane-associated cytokinetic ring that defines the division plane in bacterial and archaeal cells. In the temperature-sensitive ftsZ84 mutant of Escherichia coli, this ring assembly is impaired at the restrictive temperature causing lethal cell filamentation. Here I present genetic and morphological evidence that a 2-fold higher dosage of the division gene zipA suppresses thermosensitivity of the ftsZ84 mutant by stabilizing the labile FtsZ84 ring structure in vivo. I demonstrate that purified ZipA promotes and stabilizes protofilament assembly of both FtsZ and FtsZ84 in vitro and cosediments with the protofilaments. Furthermore, ZipA organizes FtsZ protofilaments into arrays of long bundles or sheets that probably represent the physiological organization of the FtsZ ring in bacterial cells. The N-terminal cytoplasmic domain of membrane-anchored ZipA contains sequence elements that resemble the microtubule-binding signature motifs in eukaryotic Tau, MAP2 and MAP4 proteins. It is postulated that the MAP-Tau-homologous motifs in ZipA mediate its binding to FtsZ, and that FtsZ-ZipA interaction represents an ancient prototype of the protein-protein interaction that enables MAPs to suppress microtubule catastrophe and/or to promote rescue.     

Dynamic Interaction of the Escherichia coli Cell Division ZipA and FtsZ Proteins Evidenced in Nanodiscs

The full-length ZipA protein from Escherichia coli, one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein, has been incorporated in single copy into nanodiscs formed by a membrane scaffold protein encircling an E. coli phospholipid mixture. This is an acellular system that reproduces the assembly of part of the cell division components. ZipA contained in nanodiscs (Nd-ZipA) retains the ability to interact with FtsZ oligomers and with FtsZ polymers. Interactions with FtsZ occur at similar strengths as those involved in the binding of the soluble form of ZipA, lacking the transmembrane region, suggesting that the transmembrane region of ZipA has little influence on the formation of the ZipA·FtsZ complex. Peptides containing partial sequences of the C terminus of FtsZ compete with FtsZ polymers for binding to Nd-ZipA. The affinity of Nd-ZipA for the FtsZ polymer formed with GTP or GMPCPP (a slowly hydrolyzable analog of GTP) is moderate (micromolar range) and of similar magnitude as for FtsZ-GDP oligomers. Polymerization does not stabilize the binding of FtsZ to ZipA. This supports the role of ZipA as a passive anchoring device for the proto-ring with little implication, if any, in the regulation of its assembly. Furthermore, it indicates that the tethering of FtsZ to the membrane shows sufficient plasticity to allow for its release from noncentral regions of the cytoplasmic membrane and its subsequent relocation to midcell when demanded by the assembly of a division ring.     

Genetic analysis of the Escherichia coli FtsZ.ZipA interaction in the yeast two-hybrid system. Characterization of FtsZ residues essential for the interactions with ZipA and with FtsA

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division in Escherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZ(D373G)) identified eight different changes at two residues within this sequence. In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZ(D373G) failed to interact. Two mutant proteins examined restored this interaction significantly. In vivo, the alleles tested are significantly more toxic than the wild type ftsZ and cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZ(D373G), no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.     

The straight and curved conformation of FtsZ protofilaments-evidence for rapid exchange of GTP into the curved protofilament

Bacterial cell division protein FtsZ assembles into protofilaments, which can adopt a straight or curved conformation, similar to its eukaryotic homolog, tubulin. The straight protofilaments can assemble into sheets with a lattice similar to the microtubule wall. The curved protofilaments can form rings when adsorbed to a lipid monolayer, but in solution they form helices. 4 helices assemble together to make a tube, the characteristic polymer of the curved protofilament. GTP favors the straight conformation, while GDP favors the curved. We show here that addition of EDTA and GTP to tubes causes a rapid transformation to straight protofilament sheets. Apparently when the magnesium is chelated the GDP in the curved protofilaments dissociates rapidly and is replaced with GTP, and this GTP induces the transition to straight protofilaments.     

Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.     

Straight and curved conformations of FtsZ are regulated by GTP hydrolysis

FtsZ assembles in vitro into protofilaments that can adopt two conformations-the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24 degrees. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division.     

Identification of Escherichia coli ZapC (YcbW) as a component of the division apparatus that binds and bundles FtsZ polymers

Assembly of the cell division apparatus in bacteria starts with formation of the Z ring on the cytoplasmic face of the membrane. This process involves the accumulation of FtsZ polymers at midcell and their interaction with several FtsZ-binding proteins that collectively organize the polymers into a membrane-associated ring-like configuration. Three such proteins, FtsA, ZipA, and ZapA, have previously been identified in Escherichia coli. FtsA and ZipA are essential membrane-associated division proteins that help connect FtsZ polymers with the inner membrane. ZapA is a cytoplasmic protein that is not required for the fission process per se but contributes to its efficiency, likely by promoting lateral interactions between FtsZ protofilaments. We report the identification of YcbW (ZapC) as a fourth FtsZ-binding component of the Z ring in E. coli. Binding of ZapC promotes lateral interactions between FtsZ polymers and suppresses FtsZ GTPase activity. This and additional evidence indicate that, like ZapA, ZapC is a nonessential Z-ring component that contributes to the efficiency of the division process by stabilizing the polymeric form of FtsZ.     

Recruitment of ZipA to the division site by interaction with FtsZ

ZipA is an essential cell division protein in Escherichia coli that is recruited to the division site early in the division cycle. As it is anchored to the membrane and interacts with FtsZ, it is a candidate for tethering FtsZ filaments to the membrane during the formation of the Z ring. In this study, we have investigated the requirements for ZipA localization to the division site. ZipA requires FtsZ, but not FtsA or FtsI, to be localized, indicating that it is recruited by FtsZ. Consistent with this, apparently normal Z rings are formed in the absence of ZipA. The interaction between FtsZ and ZipA occurs through their carboxy-terminal domains. Although a MalE-ZipA fusion binds to FtsZ filaments, it does not affect the GTPase activity or dynamics of the filaments. These results are consistent with ZipA acting after Z ring formation, possibly to link the membrane to FtsZ filaments during invagination of the septum.     

FtsZ and the division of bacterial cell

In this review we have tried to describe proteins and supermolecular structures which take part in the division of bacterial cell. The principal cell division protein of the most of prokaryotes is FtsZ, a homologue of eukaryotic tubulin. FtsZ just as tubulin is capable to bind and hydrolyze GTP. The division of bacterial cell begins with forming of so called divisome. The basis of such divisome is a contractile ring (Z ring); the ring encircles the cell about midcell. Z ring consists of a bundle of laterally bound protofilaments, which have been formed as a result of FtsZ polymerization. Z ring is rigidly bounded to cytozolic side of inner membrane with participation of FtsA, ZipA, FtsW and many other cell division proteins of divisome. The ring directs the process of cytokinesis transmitting power of constriction to membrane. Primary structures of members of the family of prokaryotic FtsZs differ from eukaryotic tubulines significantly except the region, where the site of GTP binding is placed. There is high degree of homology between structures of these proteins in the region. FtsZ is one of the most conservative proteins in prokaryotes, but ftsZ genes have not been found in completely sequenced genomes of several species of microorganisms. There are 2 species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea among them. So these organisms divide without FtsZ. There are many open reading frames which encode proteins with unknown functions in genomes of U. parvum and M. mobile. The comparison of primary structures of these hypothetical proteins with structures of cell division proteins did not allow researchers to find similar proteins among them. We suppose that the process of cell division of these organisms should recruit proteins with function similar to FtsZ and having homologous with FtsZ or other cell division proteins spatial structures.     

The C‐terminal linker of Escherichia coli FtsZ functions as an intrinsically disordered peptide

The tubulin homologue FtsZ provides the cytoskeletal framework and constriction force for bacterial cell division. FtsZ has an ～ 50‐amino‐acid (aa) linker between the protofilament‐forming globular domain and the C‐terminal (Ct) peptide that binds FtsA and ZipA, tethering FtsZ to the membrane. This Ct‐linker is widely divergent across bacterial species and thought to be an intrinsically disordered peptide (IDP). We confirmed that the Ct‐linkers from three bacterial species behaved as IDPs in vitro by circular dichroism and trypsin proteolysis. We made chimeras, swapping the Escherichia coli linker for Ct‐linkers from other bacteria, and even for an unrelated IDP from human α‐adducin. Most substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter. With few exceptions, almost any sequence appears to work. Length, however, was important: IDPs shorter than 43 or longer than 95 aa had compromised or no function. We conclude that the Ct‐linker functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA/ZipA at the membrane. Modelling the Ct‐linker as a worm‐like chain, we predict that it functions as a stiff entropic spring linking the bending protofilaments to the membrane.     

SepF increases the assembly and bundling of FtsZ polymers and stabilizes FtsZ protofilaments by binding along its length

SepF (Septum Forming) protein has been recently identified through genetic studies, and it has been suggested to be involved in the division of Bacillus subtilis cells. We have purified functional B. subtilis SepF from the inclusion bodies overexpressed in Escherichia coli. Far-UV circular dichroism and fluorescence spectroscopic analysis involving the extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid suggested that the purified SepF had characteristics of folded proteins. SepF was found to promote the assembly and bundling of FtsZ protofilaments using three complimentary techniques, namely 90 degrees light scattering, sedimentation, and transmission electron microscopy. SepF also decreased the critical concentration of FtsZ assembly, prevented the dilution-induced disassembly of FtsZ protofilaments, and suppressed the GTPase activity of FtsZ. Further, thick bundles of FtsZ protofilaments were observed using fluorescein isothiocyanate-labeled SepF (FITC-SepF). Interestingly, FITC-SepF was found to be uniformly distributed along the length of the FtsZ protofilaments, suggesting that SepF copolymerizes with FtsZ. SepF formed a stable complex with FtsZ, as evident from the gel filtration analysis. Using a C-terminal tail truncated FtsZ (FtsZDelta16) and a C-terminal synthetic peptide of B. subtilis FtsZ (366-382); we provided evidence indicating that SepF binds primarily to the C-terminal tail of FtsZ. The present work in concert with the available in vivo data support a model in which SepF plays an important role in regulating the assembly dynamics of the divisome complex; therefore, it may have an important role in bacterial cell division.     

FtsZ Polymers Tethered to the Membrane by ZipA Are Susceptible to Spatial Regulation by Min Waves

Bacterial cell division is driven by an FtsZ ring in which the FtsZ protein localizes at mid-cell and recruits other proteins, forming a divisome. In Escherichia coli, the first molecular assembly of the divisome, the proto-ring, is formed by the association of FtsZ polymers to the cytoplasmic membrane through the membrane-tethering FtsA and ZipA proteins. The MinCDE system plays a major role in the site selection of the division ring because these proteins oscillate from pole to pole in such a way that the concentration of the FtsZ-ring inhibitor, MinC, is minimal at the cell center, thus favoring FtsZ assembly in this region. We show that MinCDE drives the formation of waves of FtsZ polymers associated to bilayers by ZipA, which propagate as antiphase patterns with respect to those of Min as revealed by confocal fluorescence microscopy. The emergence of these FtsZ waves results from the displacement of FtsZ polymers from the vicinity of the membrane by MinCD, which efficiently competes with ZipA for the C-terminal region of FtsZ, a central hub for multiple interactions that are essential for division. The coupling between FtsZ polymers and Min is enhanced at higher surface densities of ZipA or in the presence of crowding agents that favor the accumulation of FtsZ polymers near the membrane. The association of FtsZ polymers to the membrane modifies the response of FtsZ to Min, and comigrating Min-FtsZ waves are observed when FtsZ is free in solution and not attached to the membrane by ZipA. Taken together, our findings show that the dynamic Min patterns modulate the spatial distribution of FtsZ polymers in controlled minimal membranes. We propose that ZipA plays an important role in mid-cell recruitment of FtsZ orchestrated by MinCDE.     

Tubulin-like protofilaments in Ca2+-induced FtsZ sheets

The 40 kDa protein FtsZ is a major septum-forming component of bacterial cell division. Early during cytokinesis at midcell, FtsZ forms a cytokinetic ring that constricts as septation progresses. FtsZ has a high propensity to polymerize in vitro into various structures, including sheets and filaments, in a GTP-dependent manner. Together with limited sequence homology, the occurrence of the tubulin signature motif in FtsZ and a similar three-dimensional structure, this leads to the conclusion that FtsZ is the bacterial tubulin homologue. We have polymerized FtsZ1 from Methanococcus jannaschii in the presence of millimolar concentrations of Ca2+ ions to produce two-dimensional crystals of plane group P2221. Most of the protein precipitates and forms filaments approximately 23.0 nm in diameter. A three-dimensional reconstruction of tilted micrographs of FtsZ sheets in negative stain between 0 and 60 degrees shows protofilaments of FtsZ running along the sheet axis. Pairs of parallel FtsZ protofilaments associate in an antiparallel fashion to form a two-dimensional sheet. The antiparallel arrangement is believed to generate flat sheets instead of the curved filaments seen in other FtsZ polymers. Together with the subunit spacing along the protofilament axis, a fitting of the FtsZ crystal structure into the reconstruction suggests a protofilamant structure very similar to that of tubulin protofilaments.     

Interaction between cell division proteins FtsE and FtsZ

FtsE and FtsX, which are widely conserved homologs of ABC transporters and interact with each other, have important but unknown functions in bacterial cell division. Coimmunoprecipitation of Escherichia coli cell extracts revealed that a functional FLAG-tagged version of FtsE, the putative ATP-binding component, interacts with FtsZ, the bacterial tubulin homolog required to assemble the cytokinetic Z ring and recruit the components of the divisome. This interaction is independent of FtsX, the predicted membrane component of the ABC transporter, which has been shown previously to interact with FtsE. The interaction also occurred independently of FtsA or ZipA, two other E. coli cell division proteins that interact with FtsZ. In addition, FtsZ copurified with FLAG-FtsE. Surprisingly, the conserved C-terminal tail of FtsZ, which interacts with other cell division proteins, such as FtsA and ZipA, was dispensable for interaction with FtsE. In support of a direct interaction with FtsZ, targeting of a green fluorescent protein (GFP)-FtsE fusion to Z rings required FtsZ, but not FtsA. Although GFP-FtsE failed to target Z rings in the absence of ZipA, its localization was restored in the presence of the ftsA* bypass suppressor, indicating that the requirement for ZipA is indirect. Coexpression of FLAG-FtsE and FtsX under certain conditions resulted in efficient formation of minicells, also consistent with an FtsE-FtsZ interaction and with the idea that FtsE and FtsX regulate the activity of the divisome.     

The Kil Peptide of Bacteriophage λ Blocks Escherichia coli Cytokinesis via ZipA-Dependent Inhibition of FtsZ Assembly

Assembly of the essential, tubulin-like FtsZ protein into a ring-shaped structure at the nascent division site determines the timing and position of cytokinesis in most bacteria and serves as a scaffold for recruitment of the cell division machinery. Here we report that expression of bacteriophage λ kil, either from a resident phage or from a plasmid, induces filamentation of Escherichia coli cells by rapid inhibition of FtsZ ring formation. Mutant alleles of ftsZ resistant to the Kil protein map to the FtsZ polymer subunit interface, stabilize FtsZ ring assembly, and confer increased resistance to endogenous FtsZ inhibitors, consistent with Kil inhibiting FtsZ assembly. Cells with the normally essential cell division gene zipA deleted (in a modified background) display normal FtsZ rings after kil expression, suggesting that ZipA is required for Kil-mediated inhibition of FtsZ rings in vivo. In support of this model, point mutations in the C-terminal FtsZ-interaction domain of ZipA abrogate Kil activity without discernibly altering FtsZ-ZipA interactions. An affinity-tagged-Kil derivative interacts with both FtsZ and ZipA, and inhibits sedimentation of FtsZ filament bundles in vitro. Together, these data inspire a model in which Kil interacts with FtsZ and ZipA in the cell to prevent FtsZ assembly into a coherent, division-competent ring structure. Phage growth assays show that kil⁺ phage lyse ∼30% later than kil mutant phage, suggesting that Kil delays lysis, perhaps via its interaction with FtsZ and ZipA.     

Recruitment of ZipA to the Septal Ring of Escherichia coli Is Dependent on FtsZ and Independent of FtsA

Cell division in prokaryotes is mediated by the septal ring. In Escherichia coli, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.     

The bacterial cell-division protein ZipA and its interaction with an FtsZ fragment revealed by X-ray crystallography

In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, a membrane-anchored protein that binds to FtsZ, are two essential components of the septal ring structure that mediates cell division. Recent data indicate that ZipA is involved in the assembly of the ring by linking FtsZ to the cytoplasmic membrane and that the ZipA-FtsZ interaction is mediated by their C-terminal domains. We present the X-ray crystal structures of the C-terminal FtsZ-binding domain of ZipA and a complex between this domain and a C-terminal fragment of FtsZ. The ZipA domain is a six-stranded beta-sheet packed against three alpha-helices and contains the split beta-alpha-beta motif found in many RNA-binding proteins. The uncovered side of the sheet incorporates a shallow hydrophobic cavity exposed to solvent. In the complex, the 17-residue FtsZ fragment occupies this entire cavity of ZipA and binds as an extended beta-strand followed by alpha-helix. An alanine-scanning mutagenesis analysis of the FtsZ fragment was also performed, which shows that only a small cluster of the buried FtsZ side chains is critical in binding to ZipA.