The structure of the cytoplasmic domain of EpsL, an inner membrane component of the type II secretion system of Vibrio cholerae: an unusual member of the actin-like ATPase superfamily

The type II secretion system (T2SS) is used by several Gram-negative bacteria for the secretion of hydrolytic enzymes and virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 so-called "Gsp proteins" spans from a regulatory ATPase in the cytoplasm, via several signal or energy transducing proteins in the inner membrane and the pseudopilins in the periplasm, to the actual pore in the outer membrane. The human pathogen Vibrio cholerae employs such an assembly, called the Eps system, for the export of its major virulence factor, cholera toxin, from its periplasm into the lumen of the gastro-intestinal tract of the host. Here, we report the atomic structure of the major cytoplasmic domain of the inner membrane-spanning EpsL protein from V. cholerae. EpsL is the binding partner of the regulatory ATPase EpsE as well as of EpsM and pseudopilins, and is therefore a critical link between the cytoplasmic and the periplasmic part of the Eps-system. The 2.7A resolution structure was determined by a combination of Se-Met multiple anomalous dispersion (MAD) and multiple isomorphous replacement with anomalous scattering (MIRAS) phasing methods. The 28kDa cytoplasmic domain of EpsL (cyto-EpsL) consists of three beta-sheet-rich domains. With domains I and III similar to the RNaseH-fold, cyto-EpsL unexpectedly shows structural homology with the superfamily of actin-like ATPases. cyto-EpsL, however, is an unusual member of this superfamily as it misses the canonical actin domains 1B and 2B, which are common yet variable in this superfamily. Moreover, cyto-EpsL has an additional domain II, which has the topology of an SHS2-fold module. Within the superfamily this fold module has been observed only for domain 1C of the cell division protein FtsA, in which it mediates protein-protein interactions. This domain II displays great flexibility and contributes to a pronounced negatively charged canyon on the surface of cyto-EpsL. Functional data as well as structural homology and sequence conservation suggest that domain II interacts with EpsE, the major cytoplasmic binding partner of EpsL.     

Z-ring-independent interaction between a subdomain of FtsA and late septation proteins as revealed by a polar recruitment assay

FtsA, a member of the ATPase superfamily that includes actin and bacterial actin homologs, is essential for cell division of Escherichia coli and is recruited to the Z ring. In turn, recruitment of later essential division proteins to the Z ring is dependent on FtsA. In a polar recruitment assay, we found that FtsA can recruit at least two late proteins, FtsI and FtsN, to the cell poles independently of Z rings. Moreover, a unique structural domain of FtsA, subdomain 1c, which is divergent in the other ATPase superfamily members, is sufficient for this recruitment but not required for the ability of FtsA to localize to Z rings. Surprisingly, targeting the 1c subdomain to the Z ring by fusing it to FtsZ could partially suppress a thermosensitive ftsA mutation. These results suggest that subdomain 1c of FtsA is a completely independent functional domain with an important role in interacting with a septation protein subassembly.     

Analysis of the interaction of FtsZ with itself, GTP, and FtsA.

The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system. The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP. Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding. These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily. The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species. FtsZ320, which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize. FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.     

Crystal structure of the putative adapter protein MTH1859

MTH1859 from Methanobacterium thermoautotrophicum is a 77 residue protein representing a conserved family of functionally uncharacterized proteins. We solved the crystal structure of MTH1859 by single wavelength anomalous diffraction phasing using selenomethionine labeled protein. MTH1859 adopts a mainly anti-parallel all-beta-fold. The beta-sheet is heavily bent to form a U-structure that is closed through a loop. The monomer structure possesses similarities to the photoreaction center (PRC) domain fold, but the protein employs a unique oligomerization scheme. Two monomers of MTH1859 occupy the asymmetric unit and dimerize in a head-to-head fashion. Crystal packing interactions identify a second protein-protein interaction interface at the MTH1859 tails which can simultaneously bind two partner molecules. These interactions lead to the formation of a honeycomb structure and suggest that the family of MTH1859-like proteins might function as adapters for protein complex assembly.     

Role of two essential domains of Escherichia coli FtsA in localization and progression of the division ring

The FtsA protein is a member of the actin superfamily that localizes to the bacterial septal ring during cell division. Deletions of domain 1C or the S12 and S13 beta-strands in domain 2B of the Escherichia coli FtsA, previously postulated to be involved in dimerization, result in partially active proteins that do not allow the normal progression of septation. The truncated FtsA protein lacking domain 1C (FtsADelta1C) localizes in correctly placed division rings, together with FtsZ and ZipA, but does not interact with other FtsA molecules in the yeast two-hybrid assay, and fails to recruit FtsQ and FtsN into the division ring. The rings containing FtsADelta1C are therefore incomplete and do not support division. The production of high levels of FtsADelta1C causes filamentation, an effect that has been reported to result as well from the imbalance between FtsA+ and FtsZ+ molecules. These data indicate that the domain 1C of FtsA participates in the interaction of the protein with other FtsA molecules and with the other proteins that are incorporated at later stages of ring assembly, and is not involved in the interaction with FtsZ and the localization of FtsA to the septal ring. The deletion of the S12-S13 strands of domain 2B generates a protein (FtsADeltaS12-13) that retains the ability to interact with FtsA+. When the mutated protein is expressed at wild-type levels, it localizes into division rings and recruits FtsQ and FtsN, but it fails to sustain septation at normal levels resulting in filamentation. A fivefold overexpression of FtsADeltaS12-13 produces short cells that have normal division rings, but also cells with polar localization of the mutated protein, and cells with rings at abnormal positions that result in the production of a fraction (15%) of small nucleoid-free cells. The S12-S13 strands of domain 2B are not essential for septation, but affect the localization of the division ring.     

发现靶向蛋白质间相互作用的小分子药物研究进展

蛋白质-蛋白质相互作用在多种细胞功能中具有重要的作用。靶向蛋白质-蛋白质相互作用已经成为新药发现的重要策略,但发现能阻断蛋白质-蛋白质相互作用的小分子药物是一个巨大的挑战。尽管如此,近年来人们还是发现了许多能调控蛋白质-蛋白质相互作用的小分子。该文主要总结了在病毒进入、细胞凋亡通路和神经退行性疾病等方面的蛋白质-蛋白质相互作用小分子抑制剂的研究进展。     

Conserved sequence motif at the C-terminus of the bacterial cell-division protein FtsA

FtsA is an essential part of the septal ring structure in bacterial cell division. Two peptide-protein interactions are known in this process: FtsA and ZipA bind the C-terminus of FtsZ, the bacterial tubulin homologue, which is the first septal component to appear at the septum. Our recent crystal structure of FtsA revealed a possible peptide binding site on FtsA and a long disordered C-terminal region. Here we show that all FtsA proteins contain a conserved 10-13 residue motif at the C-terminal end that may facilitate targeting of downstream septal components.     

Use of yeast two-hybrid system for detection ofBacillus subtilis FtsZ protein partners

Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions. Three plasmids (pGBR1, pGBR2, pGBR3) with theClaI restriction site shifted in the three possible reading frames in fusion withGAL4 activating domain were constructed. The modified plasmids were used for identification of protein partners of FtsZ fromBacillus subtilis. Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified. The protein interactions were quantified by measurements of β-galactosidase activity in yeast cells using 4-methylumbelliferyl β-d-glactopyranoside as fluorogenic substrate.     

Novel clades of the HU/IHF superfamily point to unexpected roles in the eukaryotic centrosome,chromosome partitioning,and biologic conflicts

The HU superfamily of proteins, with a unique DNA-binding mode, has been extensively studied as the primary chromosome-packaging protein of the bacterial superkingdom. Representatives also play a role in DNA-structuring during recombination events and in eukaryotic organellar genome maintenance. However, beyond these well-studied roles, little is understood of the functional diversification of this large superfamily. Using sensitive sequence and structure analysis methods we identify multiple novel clades of the HU superfamily. We present evidence that a novel eukaryotic clade prototyped by the human CCDC81 protein acquired roles beyond DNA-binding, likely in protein-protein interaction in centrosome organization and as a potential cargo-binding protein in conjunction with Dynein-VII. We also show that these eukaryotic versions were acquired via an early lateral transfer from bacteroidetes, where we predict a role in chromosome partition. This likely happened before the last eukaryotic common ancestor, pointing to potential endosymbiont contributions beyond that of the mitochondrial progenitor. Further, we show that the dramatic lineage-specific expansion of this domain in the bacteroidetes lineage primarily is linked to a functional shift related to potential recognition and preemption of genome invasive entities such as mobile elements. Remarkably, the CCDC81 clade has undergone a similar massive lineage-specific expansion within the archosaurian lineage in birds, suggesting a possible use of the HU superfamily in a similar capacity in recognition of non-self molecules even in this case.     

Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein

We studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide-dependent manner, forming long corkscrew-like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.     

The STIR-domain superfamily in signal transduction,development and immunity

We have identified a conserved sequence segment in transmembrane receptors (including SEFs, IL17Rs) and soluble factors (including CIKS/ACT1) in eukaryotes and bacteria - the SEFIR domain. This sequence domain is part of the new STIR domain superfamily comprising also the TIR domain known to mediate TIR-TIR homotypic interactions. In TOLL/IL1R-like pathways, the cytoplasmically localized TIR domain of a receptor and the TIR domain of a soluble adaptor interact physically and activate signalling. The similarity between the SEFIR and TIR domains involves the conserved boxes 1 and 2 of the TIR domain that are implicated in homotypic dimerization, but there is no sequence similarity between SEFIR domains and the TIR sequence box 3. By analogy, we suggest that SEFIR-domain proteins function as signalling components of Toll/IL-1R-similar pathways and that their SEFIR domain mediates physical protein-protein interactions between pathway components.     

The BRCA1 C-terminal domain: structure and function

The BRCA1 C-terminal region contains a duplicated globular domain termed BRCT that is found within many DNA damage repair and cell cycle checkpoint proteins. The unique diversity of this domain superfamily allows BRCT modules to interact forming homo/hetero BRCT multimers, BRCT-non-BRCT interactions, and interactions with DNA strand breaks. The sequence and functional diversity of the BRCT superfamily suggests that BRCT domains are evolutionarily convenient interaction modules.     

Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA

The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.     

Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents

DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival. There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations. However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive. Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB. Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme. Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl-N-nitro-N-nitrosoguanidine, that act independently of DNA gyrase. Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources. In contrast, GyrI is not effective against lesions induced by ultraviolet radiation. Furthermore, the expression of GyrI does not significantly alter the topology of DNA. Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged.     

Isolation, characterization and lipid-binding properties of the recalcitrant FtsA division protein from Escherichia coli

We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins could modulate FtsA-membrane interactions. Binding of FtsA to lipids and membranes is insensitive to ionic strength, indicating that a net contribution of hydrophobic interactions is involved in the association of FtsA to lipid/membrane structures.     

Binding linkage in a telomere DNA-protein complex at the ends of Oxytricha nova chromosomes

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein-protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (K(D-DNA)=1.4 nM). Another fusion protein, constructed without the C-terminal protein-protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (K(D-DNA)=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA-protein stability to protein-protein contacts at a remote site may provide a trigger point for DNA-protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase.     

A novel structure of DNA repair protein RecO from Deinococcus radiodurans

Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries. Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery. RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination. Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties. The RecO structure is totally different from the Rad52 structure. DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain. Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair.     

Determinants of Rbp1p localization in specific cytoplasmic mRNA-processing foci, P-bodies

Rbp1p, a yeast RNA-binding protein, decreases the level of mitochondrial porin mRNA by enhancing its degradation, but the intracellular location of the Rbp1p-mediated degradation complex remains unknown. We show here that Rbp1p in xrn1Delta mutant yeast localizes in specific cytoplasmic foci that are known as P-bodies. The N-terminal and RNA recognition motif (RRM) 1 domains of Rbp1p are necessary but not sufficient for its localization in P bodies. Rbp1p forms oligomers through its C-terminal domain in vivo; N-terminal-delete, or RRM1-mutated Rbp1p can be more efficiently recruited to P-bodies in an xrn1Delta strain, expressing a full-length Rbp1p. Although POR1 mRNA is localized to P bodies in an xrn1Delta strain, this localization does not depend on Rbp1p. Decapping activator Dhh1p directly interacts with Rbp1p. However, the recruitment of Rbp1p to P-bodies does not require Dhh1p or Ccr4p. In wild-type cells, Rbp1p can localize to P-bodies under glucose deprivation or treatment with KCl. In addition, Rbp1p-mediated porin mRNA decay is elicited by Xrn1p, a 5 ' to 3 ' exonuclease. These results provide new insight into the mechanism of Rbp1p function.     

Peptides that inhibit HIV-1 integrase by blocking its protein-protein interactions

HIV-1 integrase (IN) is one of the key enzymes in the viral replication cycle. It mediates the integration of viral cDNA into the host cell genome. IN activity requires interactions with several viral and cellular proteins, as well as IN oligomerization. Inhibition of IN is an important target for the development of anti-HIV therapies, but there is currently only one anti-HIV drug used in the clinic that targets IN. Several other small-molecule anti-IN drug leads are either undergoing clinical trials or in earlier stages of development. These molecules specifically inhibit one of the IN-mediated reactions necessary for successful integration. However, small-molecule inhibitors of protein-protein interactions are difficult to develop. In this review, we focus on peptides that inhibit IN. Peptides have advantages over small-molecule inhibitors of protein-protein interactions: they can mimic the structures of the binding domains within proteins, and are large enough to competitively inhibit protein-protein interactions. The development of peptides that bind IN and inhibit its protein-protein interactions will increase our understanding of the IN mode of action, and lead to the development of new drug leads, such as small molecules derived from these peptides, for better anti-HIV therapy.