Jodi L. Camberg

ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics

FtsZ is the major cytoskeletal protein in bacteria and a tubulin homologue. It polymerizes and forms a ring where constriction occurs to divide the cell. We found that FtsZ is degraded by E. coli ClpXP, an ATP-dependent protease. In vitro, ClpXP degrades both FtsZ protomers and polymers; however, polymerized FtsZ is degraded more rapidly than the monomer. Deletion analysis shows that the N-terminal domain of ClpX is important for polymer recognition and that the FtsZ C terminus contains a ClpX recognition signal. In vivo, FtsZ is turned over slower in a clpX deletion mutant compared with a WT strain. Overexpression of ClpXP results in increased FtsZ degradation and filamentation of cells. These results suggest that ClpXP may participate in cell division by modulating the equilibrium between free and polymeric FtsZ via degradation of FtsZ filaments and protomers.

Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids

EpsE is a cytoplasmic component of the type II secretion system in Vibrio cholerae. Through ATP hydrolysis and an interaction with the cytoplasmic membrane protein EpsL, EpsE supports secretion of cholera toxin across the outer membrane. In this study, we have determined the effect of the cytoplasmic domain of EpsL (cyto‐EpsL) and purified phospholipids on the ATPase activity of EpsE. Acidic phospholipids, specifically cardiolipin, bound the copurified EpsE/cyto‐EpsL complex and stimulated its ATPase activity 30–130‐fold, whereas the activity of EpsE alone was unaffected. Removal of the last 11 residues (residues 243–253) from cyto‐EpsL prevented cardiolipin binding as well as stimulation of the ATPase activity of EpsE. Further mutagenesis of the C‐terminal region of the EpsL cytoplasmic domain adjacent to the predicted transmembrane helix suggested that this region participates in fine tuning the interaction of EpsE with the cytoplasmic membrane and influences the oligomerization state of EpsE thereby stimulating its ATPase activity and promoting extracellular secretion in V. cholerae.

Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE

The type II secretion system is a macromolecular assembly that facilitates the extracellular translocation of folded proteins in gram-negative bacteria. EpsE, a member of this secretion system in Vibrio cholerae, contains a nucleotide-binding motif composed of Walker A and B boxes that are thought to participate in binding and hydrolysis of ATP and displays structural homology to other transport ATPases. Here we demonstrate that purified EpsE is an Mg2+-dependent ATPase and define optimal conditions for the hydrolysis reaction. EpsE displays concentration-dependent activity, which may suggest that the active form is oligomeric. Size exclusion chromatography showed that the majority of purified EpsE is monomeric; however, detailed analyses of specific activities obtained following gel filtration revealed the presence of a small population of active oligomers. We further report that EpsE binds zinc through a tetracysteine motif near its carboxyl terminus, yet metal displacement assays suggest that zinc is not required for catalysis. Previous studies describing interactions between EpsE and other components of the type II secretion pathway together with these data further support the hypothesis that EpsE functions to couple energy to the type II apparatus, thus enabling secretion.

Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling

Molecular chaperones are proteins that assist the folding, unfolding, and remodeling of other proteins. In eukaryotes, heat shock protein 90 (Hsp90) proteins are essential ATP-dependent molecular chaperones that remodel and activate hundreds of client proteins with the assistance of cochaperones. In Escherichia coli, the activity of the Hsp90 homolog, HtpG, has remained elusive. To explore the mechanism of action of E. coli Hsp90, we used in vitro protein reactivation assays. We found that E. coli Hsp90 promotes reactivation of heat-inactivated luciferase in a reaction that requires the prokaryotic Hsp70 chaperone system, known as the DnaK system. An Hsp90 ATPase inhibitor, geldanamycin, inhibits luciferase reactivation demonstrating the importance of the ATP-dependent chaperone activity of E. coli Hsp90 during client protein remodeling. Reactivation also depends upon the ATP-dependent chaperone activity of the DnaK system. Our results suggest that the DnaK system acts first on the client protein, and then E. coli Hsp90 and the DnaK system collaborate synergistically to complete remodeling of the client protein. Results indicate that E. coli Hsp90 and DnaK interact in vivo and in vitro, providing additional evidence to suggest that E. coli Hsp90 and the DnaK system function together.

Uncovering a Region of Heat Shock Protein 90 Important for Client Binding in E. coli and Chaperone Function in Yeast

The heat shock protein 90 (Hsp90) family of heat shock proteins is an abundantly expressed and highly conserved family of ATP-dependent molecular chaperones. Hsp90 facilitates remodeling and activation of hundreds of proteins. In this study, we developed a screen to identify Hsp90-defective mutants in E. coli. The mutations obtained define a region incorporating residues from the middle and C-terminal domains of E. coli Hsp90. The mutant proteins are defective in chaperone activity and client binding in vitro. We constructed homologous mutations in S. cerevisiae Hsp82 and identified several that caused defects in chaperone activity in vivo and in vitro. However, the Hsp82 mutant proteins were less severely defective in client binding to a model substrate than the corresponding E. coli mutant proteins. Our results identify a region in Hsp90 important for client binding in E. coli Hsp90 and suggest an evolutionary divergence in the mechanism of client interaction by bacterial and yeast Hsp90.

The Interplay of ClpXP with the Cell Division Machinery in Escherichia coli

ClpXP is a two-component protease composed of ClpX, an ATP-dependent chaperone that recognizes and unfolds specific substrates, and ClpP, a serine protease. One ClpXP substrate in Escherichia coli is FtsZ, which is essential for cell division. FtsZ polymerizes and forms the FtsZ ring at midcell, where division occurs. To investigate the role of ClpXP in cell division, we examined the effects of clpX and clpP deletions in several strains that are defective for cell division. Together, our results suggested that ClpXP modulates cell division through degradation of FtsZ and possibly other cell division components that function downstream of FtsZ ring assembly. In the ftsZ84 strain, which is temperature sensitive for filamentation due to a mutation in ftsZ, we observed that deletion of clpX or clpP suppresses filamentation and reduces FtsZ84 degradation. These results are consistent with ClpXP playing a role in cell division by modulating the level of FtsZ through degradation. In another division-defective strain, ΔminC, the additional deletion of clpX or clpP delays cell division and exacerbates filamentation. Our results demonstrate that ClpXP modulates division in cells lacking MinC by a mechanism that requires ATP-dependent degradation. However, antibiotic chase experiments in vivo indicate that FtsZ degradation is slower in the ΔminC strain than in the wild type, suggesting there may be another cell division component degraded by ClpXP. Taken together these studies suggest that ClpXP may degrade multiple cell division proteins, thereby modulating the precise balance of the components required for division.

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.

The bacterial cell division regulators MinD and MinC form polymers in the presence of nucleotide

The Min system of proteins, consisting of MinC, MinD and MinE, is essential for normal cell division in Escherichia coli. MinC forms a polar gradient to restrict placement of the division septum to midcell. MinC localization occurs through a direct interaction with MinD, a membrane‐associating Par‐like ATPase. MinE stimulates ATP hydrolysis by MinD, thereby releasing MinD from the membrane. Here, we show that MinD forms polymers with MinC and ATP without the addition of phospholipids. The topological regulator MinE induces disassembly of MinCD polymers. Two MinD mutant proteins, MinD(K11A) and MinD(ΔMTS15), are unable to form polymers with MinC.

Zinc coordination is essential for the function and activity of the type II secretion ATPase EpsE

The type II secretion system Eps in Vibrio cholerae promotes the extracellular transport of cholera toxin and several hydrolytic enzymes and is a major virulence system in many Gram‐negative pathogens which is structurally related to the type IV pilus system. The cytoplasmic ATPase EpsE provides the energy for exoprotein secretion through ATP hydrolysis. EpsE contains a unique metal‐binding domain that coordinates zinc through a tetracysteine motif (CXXCX29CXXC), which is also present in type IV pilus assembly but not retraction ATPases. Deletion of the entire domain or substitution of any of the cysteine residues that coordinate zinc completely abrogates secretion in an EpsE‐deficient strain and has a dominant negative effect on secretion in the presence of wild‐type EpsE. Consistent with the in vivo data, chemical depletion of zinc from purified EpsE hexamers results in loss of in vitro ATPase activity. In contrast, exchanging the residues between the two dicysteines with those from the homologous ATPase XcpR from Pseudomonas aeruginosa does not have a significant impact on EpsE. These results indicate that, although the individual residues in the metal‐binding domain are generally interchangeable, zinc coordination is essential for the activity and function of EpsE.

FtsA reshapes membrane architecture and remodels the Z-ring in Escherichia coli : FtsA reshapes membrane architecture

Cell division in prokaryotes initiates with assembly of the Z‐ring at midcell, which, in Escherichia coli, is tethered to the inner leaflet of the cytoplasmic membrane through a direct interaction with FtsA, a widely conserved actin homolog. The Z‐ring is comprised of polymers of tubulin‐like FtsZ and has been suggested to provide the force for constriction. Here, we demonstrate that FtsA exerts force on membranes causing redistribution of membrane architecture, robustly hydrolyzes ATP and directly engages FtsZ polymers in a reconstituted system. Phospholipid reorganization by FtsA occurs rapidly and is mediated by insertion of a C‐terminal membrane targeting sequence (MTS) into the bilayer and further promoted by a nucleotide‐dependent conformational change relayed to the MTS. FtsA also recruits FtsZ to phospholipid vesicles via a direct interaction with the FtsZ C‐terminus and regulates FtsZ assembly kinetics. These results implicate the actin homolog FtsA in establishment of a Z‐ring scaffold, while directly remodeling the membrane and provide mechanistic insight into localized cell wall remodeling, invagination and constriction at the onset of division.