Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease.

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.IMPORTANCE Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in Vibrio cholerae as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other Vibrio species and close relatives, and this may be why inactivation of the T2SS in species such as Vibrio vulnificus, Vibrio sp. strain 60, and Aeromonas hydrophila also results in a pleiotropic effect on their outer membrane assembly and integrity.

The crystal structure of the drug target Mycobacterium tuberculosis methionyl-tRNA synthetase in complex with a catalytic intermediate.

Mycobacterium tuberculosis is a pathogenic bacterial infectious agent that is responsible for approximately 1.5 million human deaths annually. Current treatment requires the long-term administration of multiple medicines with substantial side effects. Lack of compliance, together with other factors, has resulted in a worrisome increase in resistance. New treatment options are therefore urgently needed. Here, the crystal structure of methionyl-tRNA synthetase (MetRS), an enzyme critical for protein biosynthesis and therefore a drug target, in complex with its catalytic intermediate methionyl adenylate is reported. Phenylalanine 292 of the M. tuberculosis enzyme is in an `out' conformation and barely contacts the adenine ring, in contrast to other MetRS structures where ring stacking occurs between the adenine and a protein side-chain ring in the `in' conformation. A comparison with human cytosolic MetRS reveals substantial differences in the active site as well as regarding the position of the connective peptide subdomain 1 (CP1) near the active site, which bodes well for arriving at selective inhibitors. Comparison with the human mitochondrial enzyme at the amino-acid sequence level suggests that arriving at inhibitors with higher affinity for the mycobacterial enzyme than for the mitochondrial enzyme might be achievable.

Leishmania donovani tyrosyl-tRNA synthetase structure in complex with a tyrosyl adenylate analog and comparisons with human and protozoan counterparts.

The crystal structure of Leishmania donovani tyrosyl-tRNA synthetase (LdTyrRS) in complex with a nanobody and the tyrosyl adenylate analog TyrSA was determined at 2.75 Å resolution. Nanobodies are the variable domains of camelid heavy chain-only antibodies. The nanobody makes numerous crystal contacts and in addition reduces the flexibility of a loop of LdTyrRS. TyrSA is engaged in many interactions with active site residues occupying the tyrosine and adenine binding pockets. The LdTyrRS polypeptide chain consists of two pseudo-monomers, each consisting of two domains. Comparing the two independent chains in the asymmetric unit reveals that the two pseudo-monomers of LdTyrRS can bend with respect to each other essentially as rigid bodies. This flexibility might be useful in the positioning of tRNA for catalysis since both pseudo-monomers in the LdTyrRS chain are needed for charging tRNATyr. An "extra pocket" (EP) appears to be present near the adenine binding region of LdTyrRS. Since this pocket is absent in the two human homologous enzymes, the EP provides interesting opportunities for obtaining selective drugs for treating infections caused by L. donovani, a unicellular parasite causing visceral leishmaniasis, or kala azar, which claims 20,000 to 30,000 deaths per year. Sequence and structural comparisons indicate that the EP is a characteristic which also occurs in the active site of several other important pathogenic protozoa. Therefore, the structure of LdTyrRS could inspire the design of compounds useful for treating several different parasitic diseases.

Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity.

The type II secretion system (T2SS), a multiprotein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multidomain T2SS assembly ATPase GspE(EpsE) had not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspE(EpsE) are obtained when fused to Hcp1 as an assistant hexamer, as shown with native mass spectrometry. The enzymatic activity of the GspE(EpsE)-Hcp1 fusions is ∼20 times higher than that of a GspE(EpsE) monomer, indicating that increasing the local concentration of GspE(EpsE) by the fusion strategy was successful. Crystal structures of GspE(EpsE)-Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspE(EpsE) with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspE(EpsE)-Hcp1 fusions suggest that even further variability in GspE(EpsE) hexamer architecture is likely.

The compact conformation of the Plasmodium knowlesi myosin tail interacting protein MTIP in complex with the C-terminal helix of myosin A.

The myosin motor of the malaria parasite's invasion machinery moves over actin fibers while it is making critical contacts with the myosin-tail interacting protein (MTIP). Previously, in a "compact" Plasmodium falciparum MTIP•MyoA complex, MTIP domains 2 (D2) and 3 (D3) make contacts with the MyoA helix, and the central helix is kinked, but in an "extended" Plasmodium knowlesi MTIP•MyoA complex only D3 interacts with the MyoA helix, and the central helix is fully extended. Here we report the crystal structure of the compact P. knowlesi MTIP•MyoA complex. It appears that, depending on the pH, P. knowlesi MTIP can adopt either the compact or the extended conformation to interact with MyoA. Only at pH values above ~7.0, can key hydrogen bonds can be formed by the imidazole group of MyoA His810 with an aspartate carboxylate from the hinge of MTIP and a lysine amino group of MyoA simultaneously.

The structure of the D3 domain of Plasmodium falciparum myosin tail interacting protein MTIP in complex with a nanobody.

Apicomplexan parasites enter host cells by many sophisticated steps including use of an ATP-powered invasion machinery. The machinery consists of multiple proteins, including a special myosin (MyoA) which moves along an actin fiber and which is connected to the myosin tail interaction protein (MTIP). Here we report a crystal structure of the major MyoA-binding domain (D3) of Plasmodium falciparum MTIP in complex with an anti-MTIP nanobody. In this complex, the MyoA-binding groove in MTIP-D3 is considerably less accessible than when occupied by the MyoA helix, due to a shift of two helices. The nanobody binds to an area slightly overlapping with the MyoA binding groove, covering a hydrophobic region next to the groove entrance. This provides a new avenue for arriving at compounds interfering with the invasion machinery since small molecules binding simultaneously to the nanobody binding site and the adjacent MyoA binding groove would prevent MyoA binding by MTIP.

Structural and functional studies on the interaction of GspC and GspD in the type II secretion system.

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains. Three different crystal structures of the homology region domain of GspC (GspC(HR)) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC(HR) adopts an all-ß topology. N-terminal ß-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC(HR)-GspD(N0) interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.

Structures of a key interaction protein from the Trypanosoma brucei editosome in complex with single domain antibodies.

Several major global diseases are caused by single-cell parasites called trypanosomatids. These organisms exhibit many unusual features including a unique and essential U-insertion/deletion RNA editing process in their single mitochondrion. Many key RNA editing steps occur in ∼20S editosomes, which have a core of 12 proteins. Among these, the "interaction protein" KREPA6 performs a central role in maintaining the integrity of the editosome core and also binds to ssRNA. The use of llama single domain antibodies (VHH domains) accelerated crystal growth of KREPA6 from Trypanosoma brucei dramatically. All three structures obtained are heterotetramers with a KREPA6 dimer in the center, and one VHH domain bound to each KREPA6 subunit. Two of the resultant heterotetramers use complementarity determining region 2 (CDR2) and framework residues to form a parallel pair of beta strands with KREPA6 - a mode of interaction not seen before in VHH domain-protein antigen complexes. The third type of VHH domain binds in a totally different manner to KREPA6. Intriguingly, while KREPA6 forms tetramers in solution adding either one of the three VHH domains results in the formation of a heterotetramer in solution, in perfect agreement with the crystal structures. Biochemical solution studies indicate that the C-terminal tail of KREPA6 is involved in the dimerization of KREPA6 dimers to form tetramers. The implications of these crystallographic and solution studies for possible modes of interaction of KREPA6 with its many binding partners in the editosome are discussed.

Calcium is essential for the major pseudopilin in the type 2 secretion system.

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.

The closed MTIP-myosin A-tail complex from the malaria parasite invasion machinery.

The Myosin A-tail interacting protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side-chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side-chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites.

NADP+ expels both the co-factor and a substrate analog from the Mycobacterium tuberculosis ThyX active site: opportunities for anti-bacterial drug design.

The novel flavin-dependent thymidylate synthase, ThyX, is absent in humans but several pathogenic bacteria depend exclusively on ThyX activity to synthesize thymidylate. Reduction of the enzyme-bound FAD by NADPH is suggested to be the critical first step in ThyX catalysis. We soaked Mycobacterium tuberculosis ThyX-FAD-BrdUMP ternary complex crystals in a solution containing NADP+ to gain structural insights into the reductive step of the catalytic cycle. Surprisingly, the NADP+ displaced both FAD and BrdUMP from the active site. In the resultant ThyX-NADP+ binary complex, the AMP moiety is bound in a deep pocket similar to that of the same moiety of FAD in the ternary complex, while the nicotinamide part of NADP+ is engaged in a limited number of contacts with ThyX. The additional 2'-phosphate group attached to the AMP ribose of NADP+ could be accommodated with minor rearrangement of water molecules. The newly introduced 2'-phosphate groups are engaged in water-mediated interactions across the non-crystallographic 2-fold axis of the ThyX tetramer, suggesting possibilities for design of high-affinity bivalent inhibitors of this intriguing enzyme.

Structure of the MTIP-MyoA complex, a key component of the malaria parasite invasion motor.

The causative agents of malaria have developed a sophisticated machinery for entering multiple cell types in the human and insect hosts. In this machinery, a critical interaction occurs between the unusual myosin motor MyoA and the MyoA-tail Interacting Protein (MTIP). Here we present one crystal structure that shows three different conformations of Plasmodium MTIP, one of these in complex with the MyoA-tail, which reveal major conformational changes in the C-terminal domain of MTIP upon binding the MyoA-tail helix, thereby creating several hydrophobic pockets in MTIP that are the recipients of key hydrophobic side chains of MyoA. Because we also show that the MyoA helix is able to block parasite growth, this provides avenues for designing antimalarials.

Structural basis for UTP specificity of RNA editing TUTases from Trypanosoma brucei.

Trypanosomatids are pathogenic protozoa that undergo a unique form of post-transcriptional RNA editing that inserts or deletes uridine nucleotides in many mitochondrial pre-mRNAs. Editing is catalyzed by a large multiprotein complex, the editosome. A key editosome enzyme, RNA editing terminal uridylyl transferase 2 (TUTase 2; RET2) catalyzes the uridylate addition reaction. Here, we report the 1.8 A crystal structure of the Trypanosoma brucei RET2 apoenzyme and its complexes with uridine nucleotides. This structure reveals that the specificity of the TUTase for UTP is determined by a crucial water molecule that is exquisitely positioned by the conserved carboxylates D421 and E424 to sense a hydrogen atom on the N3 position of the uridine base. The three-domain structure also unveils a unique domain arrangement not seen before in the nucleotidyltansferase superfamily, with a large domain insertion between the catalytic aspartates. This insertion is present in all trypanosomatid TUTases. We also show that TbRET2 is essential for survival of the bloodstream form of the parasite and therefore is a potential target for drug therapy.

Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0A resolution.

A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M.tuberculosis ThyX (MtbThyX) enzyme complements the E.coli chi2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.

An improved crystal form of Plasmodium falciparum peptide deformylase.

An altered version of peptide deformylase from Plasmodium falciparum (PfPDF), the organism that causes the most devastating form of malaria, has been cocrystallized with a synthesized inhibitor that has submicromolar affinity for its target protein. The structure is solved at 2.2 A resolution, an improvement over the 2.8 A resolution achieved during the structural determination of unliganded PfPDF. This represents the successful outcome of modifying the protein construct in order to overcome adverse crystal contacts and other problems encountered in the study of unliganded PfPDF. Two molecules of PfPDF are found in the asymmetric unit of the current structure. The active site of each monomer of PfPDF is occupied by a proteolyzed fragment of the tripeptide-like inhibitor. Unexpectedly, each PfPDF subunit is associated with two nearly complete molecules of the inhibitor, found at a protein-protein interface. This is the first structure of a eukaryotic PDF protein, a potential drug target, in complex with a ligand.

The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding.

Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-A crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches.

Crystals of peptide deformylase from Plasmodium falciparum reveal critical characteristics of the active site for drug design.

Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.