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.     

Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction

A fluorescence polarization competition assay has been developed to screen for inhibitors of the Escherichia coli FtsZ/ZipA protein-protein interaction. A previously published X-ray costructure demonstrated that a 17-amino-acid peptide, corresponding to FtsZ C-terminal residues 367-383 (FtsZ(367-383)), interacts with the C-terminal FtsZ binding domain of ZipA (ZipA(185-328)). Phage display was employed to identify a unique but related peptide which when further modified and labeled was shown to have a higher affinity to ZipA(185-328) than the FtsZ(367-383) peptide and binds to the same site. This peptide had a six fold increase in fluorescence polarization upon binding to ZipA(185-328) compared to a two fold increase for the FtsZ(367-383) fluorophore. As a result, assay parameters using the phage display peptide were further optimized and adapted for the high-throughput screen. A high-throughput screen of 250,000 compounds identified 29 hits with inhibition equal to or greater than 30% at 50 microg/ml. An X-ray costructure of a promising small molecule in this library complexed with ZipA(185-328) (KI=12 microM) revealed that the compound binds to the same hydrophobic pocket as the FtsZ(367-383) peptide.     

Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes

Absolute binding free energy calculations and free energy decompositions are presented for the protein-protein complexes H-Ras/C-Raf1 and H-Ras/RalGDS. Ras is a central switch in the regulation of cell proliferation and differentiation. In our study, we investigate the capability of the molecular mechanics (MM)-generalized Born surface area (GBSA) approach to estimate absolute binding free energies for the protein-protein complexes. Averaging gas-phase energies, solvation free energies, and entropic contributions over snapshots extracted from trajectories of the unbound proteins and the complexes, calculated binding free energies (Ras-Raf: -15.0(+/-6.3)kcal mol(-1); Ras-RalGDS: -19.5(+/-5.9)kcal mol(-1)) are in fair agreement with experimentally determined values (-9.6 kcal mol(-1); -8.4 kcal mol(-1)), if appropriate ionic strength is taken into account. Structural determinants of the binding affinity of Ras-Raf and Ras-RalGDS are identified by means of free energy decomposition. For the first time, computationally inexpensive generalized Born (GB) calculations are applied in this context to partition solvation free energies along with gas-phase energies between residues of both binding partners. For selected residues, in addition, entropic contributions are estimated by classical statistical mechanics. Comparison of the decomposition results with experimentally determined binding free energy differences for alanine mutants of interface residues yielded correlations with r(2)=0.55 and 0.46 for Ras-Raf and Ras-RalGDS, respectively. Extension of the decomposition reveals residues as far apart as 25A from the binding epitope that can contribute significantly to binding free energy. These "hotspots" are found to show large atomic fluctuations in the unbound proteins, indicating that they reside in structurally less stable regions. Furthermore, hotspot residues experience a significantly larger-than-average decrease in local fluctuations upon complex formation. Finally, by calculating a pair-wise decomposition of interactions, interaction pathways originating in the binding epitope of Raf are found that protrude through the protein structure towards the loop L1. This explains the finding of a conformational change in this region upon complex formation with Ras, and it may trigger a larger structural change in Raf, which is considered to be necessary for activation of the effector by Ras.     

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 conserved C-terminal tail of FtsZ is required for the septal localization and division inhibitory activity of MinCC/MinD

The Escherichia coli Min system contributes to spatial regulation of cytokinesis by preventing assembly of the Z ring away from midcell. MinC is a cell division inhibitor whose activity is spatially regulated by MinD and MinE. MinC has two functional domains of similar size, both of which have division inhibitory activity in the proper context. However, the molecular mechanism of the inhibitory action of either domain is not very clear. Here, we report that the septal localization and division inhibitory activity of MinC^C/MinD requires the conserved C-terminal tail of FtsZ. This tail also mediates interaction with two essential division proteins, ZipA and FtsA, to link FtsZ polymers to the membrane. Overproduction of MinC^C/MinD displaces FtsA from the Z ring and eventually disrupts the Z ring, probably because it also displaces ZipA. These results support a model for the division inhibitory action of MinC/MinD. MinC/MinD binds to ZipA and FtsA decorated FtsZ polymers located at the membrane through the MinC^C/MinD–FtsZ interaction. This binding displaces FtsA and/or ZipA, and more importantly, positions MinC^N near the FtsZ polymers making it a more effective inhibitor.     

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.     

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.     

Genetic and functional analyses of the conserved C-terminal core domain of Escherichia coli FtsZ

In Escherichia coli, FtsZ is required for the recruitment of the essential cell division proteins FtsA and ZipA to the septal ring. Several C-terminal deletions of E. coli FtsZ, including one of only 12 amino acids that removes the highly conserved C-terminal core domain, failed to complement chromosomal ftsZ mutants when expressed on a plasmid. To identify key individual residues within the core domain, six highly conserved residues were replaced with alanines. All but one of these mutants (D373A) failed to complement an ftsZ chromosomal mutant. Immunoblot analysis demonstrated that whereas I374A and F377A proteins were unstable in the cell, L372A, D373A, P375A, and L378A proteins were synthesized at normal levels, suggesting that they were specifically defective in some aspect of FtsZ function. In addition, all four of the stable mutant proteins were able to localize and form rings at potential division sites in chromosomal ftsZ mutants, implying a defect in a function other than localization and multimerization. Because another proposed function of FtsZ is the recruitment of FtsA and ZipA, we tested whether the C-terminal core domain was important for interactions with these proteins. Using two different in vivo assays, we found that the 12-amino-acid truncation of FtsZ was defective in binding to FtsA. Furthermore, two point mutants in this region (L372A and P375A) showed weakened binding to FtsA. In contrast, ZipA was capable of binding to all four stable point mutants in the FtsZ C-terminal core but not to the 12-amino-acid deletion.     

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.     

Energetics of sequence-specific protein-DNA association: computational analysis of integrase Tn916 binding to its target DNA

The N-terminal domain of the bacterial integrase Tn916 specifically recognizes the 11 bp DNA target site by positioning the face of a three-stranded beta-sheet into the major groove. Binding is linked to structural adaptation. We have characterized INT-DBD binding to DNA in detail by calorimetry [Milev, S., Gorfe, A., Karshikoff, A., Clubb, R. T., Bosshard, H. R., and Jelesarov, I. (2003) Biochemistry 42, 3481-3491]. Our thermodynamic analysis has indicated that the major driving force of association is the hydrophobic effect while polar interactions contribute less. To gain more comprehensive information about the binding process, we performed a computational analysis of the binding free energy and report here the results. A hybrid molecular mechanics/continuum approach was followed. The total binding free energy is predicted with reasonable accuracy. The calculations confirm that nonpolar effects stabilize the protein-DNA complex while electrostatics opposes binding. Structural changes optimizing surface complementarity are costly in terms of energy. The energetic consequences from the replacement of nine DNA-contacting residues by alanine were investigated. The calculations correctly predict the binding affinity decrease of eight mutations and the destabilizing effect of one wild-type residue. Bulky side chains stabilize the wild-type complex through packing interactions and favorable nonpolar dehydration, but the net nonpolar energy changes do not correlate with the relative affinity loss upon mutation. Discrete protein-DNA electrostatic interactions may be net stabilizing or net destabilizing depending on the local environment. In contrast to nonpolar energy changes, the magnitude of the electrostatic free energy ranks the mutations according to the experimentally measured DeltaDeltaG. Free energy decomposition analysis from a structural perspective leads to detailed information about the thermodynamic strategy used by INT-DBD for sequence-specific DNA binding.     

Monte Carlo simulations of the peptide recognition at the consensus binding site of the constant fragment of human immunoglobulin G: the energy landscape analysis of a hot spot at the intermolecular interface

Monte Carlo simulations of molecular recognition at the consensus binding site of the constant fragment (Fc) of human immunoglobulin G (Ig) protein have been performed to analyze structural and thermodynamic aspects of binding for the 13-residue cyclic peptide DCAWHLGELVWCT. The energy landscape analysis of a hot spot at the intermolecular interface using alanine scanning and equilibrium-simulated tempering dynamics with the simplified, knowledge-based energy function has enabled the role of the protein hot spot residues in providing the thermodynamic stability of the native structure to be determined. We have found that hydrophobic interactions between the peptide and the Met-252, Ile-253, His-433, and His-435 protein residues are critical to guarantee the thermodynamic stability of the crystallographic binding mode of the complex. Binding free energy calculations, using a molecular mechanics force field and a solvation energy model, combined with alanine scanning have been conducted to determine the energetic contribution of the protein hot spot residues in binding affinity. The conserved Asn-434, Ser-254, and Tyr-436 protein residues contribute significantly to the binding affinity of the peptide-protein complex, serving as an energetic hot spot at the intermolecular interface. The results suggest that evolutionary conserved hot spot protein residues at the intermolecular interface may be partitioned in fulfilling thermodynamic stability of the native binding mode and contributing to the binding affinity of the complex.     

Discovery of novel inhibitors of the ZipA/FtsZ complex by NMR fragment screening coupled with structure-based design

ZipA is a membrane anchored protein in Escherichia coli that interacts with FtsZ, a homolog of eukaryotic tubulins, forming a septal ring structure that mediates bacterial cell division. Thus, the ZipA/FtsZ protein-protein interaction is a potential target for an antibacterial agent. We report here an NMR-based fragment screening approach which identified several hits that bind to the C-terminal region of ZipA. The screen was performed by 1H-15N HSQC experiments on a library of 825 fragments that are small, lead-like, and highly soluble. Seven hits were identified, and the binding mode of the best one was revealed in the X-ray crystal structure. Similar to the ZipA/FtsZ contacts, the driving force in the binding of the small molecule ligands to ZipA is achieved through hydrophobic interactions. Analogs of this hit were also evaluated by NMR and X-ray crystal structures of these analogs with ZipA were obtained, providing structural information to help guide the medicinal chemistry efforts.     

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.     

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.     

Molecular mechanism of the enhanced virulence of 2009 pandemic Influenza A (H1N1) virus from D222G mutation in the hemagglutinin: a molecular modeling study

D222G mutation of the hemagglutinin (HA) is of special interest because of its close association with the enhanced virulence of 2009 pandemic influenza A (H1N1) virus through the increased binding affinity to α2,3-linked sialylated glycan receptors. However, there is still a lack of detailed understanding about the molecular mechanism of this enhanced virulence. Here, molecular dynamics simulation and binding free energy calculation were performed to explore the altered glycan receptor binding mechanism of HA upon the D222G mutation by studying the interaction of one α2,3-linked sialylglycan (sequence: SIA-GAL-NAG) with the wild type and D222G mutated HA. The binding free energy calculation based on the molecular mechanics generalized Born surface area (MM-GBSA) method indicates that the D222G mutated HA has a much stronger binding affinity with the studied α2,3-linked glycan than the wild type. This is consistent with the experimental result. The increased binding free energy of D222G mutant mainly comes from the increased energy contribution of Gln223. The structural analysis proves that the altered electrostatic potential of receptor binding domain (RBD) and the increased flexibility of 220-loop are the essential reasons leading to the increased affinity of HA to α2,3-linked sialic acid glycans. The obtained results of this study have allowed a deeper understanding of the receptor recognition mechanism and the pathogenicity of influenza virus, which will be valuable to the structure-based inhibitors design targeting influenza virus entry process.     

Molecular dynamics-solvated interaction energy studies of protein-protein interactions: the MP1-p14 scaffolding complex

Using the MP1-p14 scaffolding complex from the mitogen-activated protein kinase signaling pathway as model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. Hot spots are located by virtual alanine-scanning consensus predictions over three different energy functions and two different single-structure representations of the complex. Refined binding affinity predictions for select hot-spot mutations are carried out by applying first-principle methods such as the molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) to the molecular dynamics (MD) trajectories for mutated and wild-type complexes. Here, predicted hot-spot residues were actually mutated to alanine, and crystal structures of the mutated complexes were determined. Two mutated MP1-p14 complexes were investigated, the p14(Y56A)-mutated complex and the MP1(L63A,L65A)-mutated complex. Alternative ways to generate MD ensembles for mutant complexes, not relying on crystal structures for mutated complexes, were also investigated. The SIE function, fitted on protein-ligand binding affinities, gave absolute binding affinity predictions in excellent agreement with experiment and outperformed standard MM-GBSA predictions when tested on the MD ensembles of Ras-Raf and Ras-RalGDS protein-protein complexes. For wild-type and mutant MP1-p14 complexes, SIE predictions of relative binding affinities were supported by a yeast two-hybrid assay that provided semiquantitative relative interaction strengths. Results on the MP1-mutated complex suggested that SIE predictions deteriorate if mutant MD ensembles are approximated by just mutating the wild-type MD trajectory. The SIE data on the p14-mutated complex indicated feasibility for generating mutant MD ensembles from mutated wild-type crystal structure, despite local structural differences observed upon mutation. For energetic considerations, this would circumvent costly needs to produce and crystallize mutated complexes. The sensitized protein-protein interface afforded by the p14(Y56A) mutation identified here has practical applications in screening-based discovery of first-generation small-molecule hits for further development into specific modulators of the mitogen-activated protein kinase signaling pathway.     

Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli

ZipA and FtsA are essential division proteins in Escherichia coli that are recruited to the division site by interaction with FtsZ. Utilizing a newly isolated temperature-sensitive mutation in zipA we have more fully characterized the role of ZipA. We confirmed that ZipA is not required for Z ring formation; however, we found that ZipA, like FtsA, is required for recruitment of FtsK and therefore all downstream division proteins. In the absence of FtsA or ZipA Z rings formed; however, in the absence of both, new Z rings were unable to form and preformed Z rings were destabilized. Consistent with this, we found that an FtsZ mutant unable to interact with both ZipA and FtsA was unable to assemble into Z rings. These results demonstrate that ZipA and FtsA are both required for recruitment of additional division proteins to the Z ring, but either one is capable of supporting formation and stabilization of Z rings.     

Identification of a region of FtsA required for interaction with FtsZ

The assembly of the Z ring is the earliest step in bacterial cell division. In Escherichia coli this assembly requires either FtsA or ZipA which bind to a conserved, C-terminal 17 amino acid motif in FtsZ and to the membrane. The FtsZ-ZipA interaction is well characterized; however, nothing is known about the region of FtsA involved in the interaction with FtsZ even though the FtsA-FtsZ interaction is nearly ubiquitous in Eubacteria. FtsA is proposed to bind to the membrane through its conserved C-terminal amphiphatic helix before efficiently interacting with FtsZ. Based upon this model we designed a genetic screen to identify mutants specifically impaired for the FtsA-FtsZ interaction. The mutants obtained retain the ability to be targeted to the membrane but fail to be recruited to the Z ring or interact with FtsZ in the yeast two-hybrid system. These mutants do not complement an ftsA-depletion strain. Through this approach we have identified a region of FtsA containing some invariant residues which is required for binding to FtsZ. The results support our model that FtsA is targeted to the membrane before it interacts with FtsZ and demonstrates that this interaction plays an essential role in E. coli cell division.     

Combinatorial synthesis of substituted 3-(2-indolyl)piperidines and 2-phenyl indoles as inhibitors of ZipA-FtsZ interaction

The ZipA-FtsZ protein-protein interaction is a potential target for antibacterial therapy. The design and parallel synthesis of a combinatorial library of small molecules, which target the FtsZ binding area on ZipA are described. Compounds were demonstrated to bind to the FtsZ binding domain of ZipA by HSQC NMR and to inhibit cell division in a cell elongation assay.     

Location of Dual Sites in E. coli FtsZ Important for Degradation by ClpXP; One at the C-Terminus and One in the Disordered Linker

ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. One substrate degraded by Escherichia coli ClpXP is FtsZ, an essential cell division protein. FtsZ forms polymers that assemble into a large ring-like structure, termed the Z-ring, during cell division at the site of constriction. The FtsZ monomer is composed of an N-terminal polymerization domain, an unstructured linker region and a C-terminal conserved region. To better understand substrate selection by ClpXP, we engineered FtsZ mutant proteins containing amino acid substitutions or deletions near the FtsZ C-terminus. We identified two discrete regions of FtsZ important for degradation of both FtsZ monomers and polymers by ClpXP in vitro. One region is located 30 residues away from the C-terminus in the unstructured linker region that connects the polymerization domain to the C-terminal region. The other region is near the FtsZ C-terminus and partially overlaps the recognition sites for several other FtsZ-interacting proteins, including MinC, ZipA and FtsA. Mutation of either region caused the protein to be more stable and mutation of both caused an additive effect, suggesting that both regions are important. We also observed that in vitro MinC inhibits degradation of FtsZ by ClpXP, suggesting that some of the same residues in the C-terminal site that are important for degradation by ClpXP are important for binding MinC.