The N-terminal FleQ domain of the Vibrio cholerae flagellar master regulator FlrA plays pivotal structural roles in stabilizing its active state.

In Vibrio cholerae, the master regulator FlrA controls transcription of downstream flagellar genes in a σ54 -dependent manner. However, the molecular basis of regulation by VcFlrA, which contains a phosphorylation-deficient N-terminal FleQ domain, has remained elusive. Our studies on VcFlrA, four of its constructs, and a mutant showed that the AAA+ domain of VcFlrA, with or without the linker 'L', remains in ATPase-deficient monomeric states. By contrast, the FleQ domain plays a pivotal role in promoting higher-order functional oligomers, providing the required conformation to 'L' for ATP/cyclic di-GMP (c-di-GMP) binding. The crystal structure of VcFlrA-FleQ at 2.0 Å suggests that distinct structural features of VcFlrA-FleQ presumably assist in inter-domain packing. VcFlrA at a high concentration forms ATPase-efficient oligomers when the intracellular c-di-GMP level is low. Conversely, excess c-di-GMP locks VcFlrA in a non-functional lower oligomeric state, causing repression of flagellar biosynthesis.

Dynamic phase separation of the androgen receptor and its coactivators key to regulate gene expression.

Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated in vitro by using recombinant full-length AR protein. We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.

The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP.

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

Mechanistic basis of vitamin B12 and cobinamide salvaging by the Vibrio species.

Biosynthesis of vitamin B12, which occurs through salvaging pathway or de novo synthesis, is essential for the survival and growth of bacteria. While the mechanism is known for many bacteria, it is elusive yet for diarrhea causing pathogenic bacteria Vibrio cholerae or the other Vibrio species. Sequence analysis using genome databases delineated that majority of the Vibrio species including V. cholerae contain genes required for salvaging cobalamin/cobinamide in aerobic pathway while lack the genes required for de novo synthesis of B12. Fluorescence quenching study showed that VcBtuF, the PBP of putative ABC transporter BtuF-CD of V. cholerae O395 binds cyanocobalamin and dicyanocobinamide with micromolar dissociation constants (Kd). Productive internalization of these nutrients has been established through growth assay. The crystal structure of cyanocobalamin bound VcBtuF has shown that although interactions between cyanocobalamin and VcBtuF are largely similar to E. coli BtuF, VcBtuF possesses a wider binding pocket. MD simulations indicated that in contrast to EcBtuF that executes 'open-close' movement, inter-lobe twisting is prevalent in VcBtuF. Although H70, located at the entrance of the substrate binding cleft of VcBtuF, executes swinging motion, it cannot act as 'closed gate' to retain cyanocobalamin or cobinamide in the pocket like corresponding residue W66 of EcBtuF. Rather, VcBtuF shows a distinctive phenomenon of heme binding with comparable affinity to B12. Soret shift of heme upon binding with VcBtuF pointed towards involvement of H70 in heme recognition. This may lead to a restricted B12 or cobinamide binding during abundance of heme in the periplasmic space.

Conserved and unique features of the fission yeast core Atg1 complex.

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.

Structure and dynamics of Type III periplasmic proteins VcFhuD and VcHutB reveal molecular basis of their distinctive ligand binding properties.

Molecular mechanisms of xenosiderophore and heme acquisitions using periplasmic binding protein (PBP) dependent ATP-binding cassette transporters to scavenge the essential nutrient iron are elusive yet in Vibrio cholerae. Our current study delineates the structures, dynamics and ligand binding properties of two Type III PBPs of V. cholerae, VcFhuD and VcHutB. Through crystal structures and fluorescence quenching studies we demonstrate unique features of VcFhuD to bind both hydroxamate and catecholate type xenosiderophores. Like E. coli FhuD, VcFhuD binds ferrichrome and ferri-desferal using conserved Tryptophans and R102. However, unlike EcFhuD, slightly basic ligand binding pocket of VcFhuD could favour ferri-enterobactin binding with plausible participation of R203, along with R102, like it happens in catecholate binding PBPs. Structural studies coupled with spectrophotometric and native PAGE analysis indicated parallel binding of two heme molecules to VcHutB in a pH dependent manner, while mutational analysis established the relative importance of Y65 and H164 in heme binding. MD simulation studies exhibited an unforeseen inter-lobe swinging motion in Type III PBPs, magnitude of which is inversely related to the packing of the linker helix with its neighboring helices. Small inter-lobe movement in VcFhuD or dramatic twisting in VcHutB is found to influence ligand binding.

Purification, crystallization and preliminary X-ray analysis of the periplasmic haem-binding protein HutB from Vibrio cholerae.

The mechanism of haem transport across the inner membrane of pathogenic bacteria is currently insufficiently understood at the molecular level and no information is available for this process in Vibrio cholerae. To obtain structural insights into the periplasmic haem-binding protein HutB from V. cholerae (VcHutB), which is involved in haem transport through the HutBCD haem-transport system, at the atomic level, VcHutB was cloned, overexpressed and crystallized using 1.6 M ammonium sulfate as a precipitant at pH 7.0. X-ray diffraction data were collected to 2.4 Šresolution on the RRCAT PX-BL-21 beamline at the Indus-2 synchrotron, Indore, India. The crystals belonged to space group P43212, with unit-cell parameters a = b = 62.88, c = 135.8 Å. Matthews coefficient calculations indicated the presence of one monomer in the asymmetric unit, with an approximate solvent content of 45.02%. Molecular-replacement calculations with Phaser confirmed the presence of a monomer in the asymmetric unit.

Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.

Bacterial enhancer-binding proteins (bEBPs) oligomerize through AAA(+) domains and use ATP hydrolysis-driven energy to isomerize the RNA polymerase-σ(54) complex during transcriptional initiation. Here, we describe the first structure of the central AAA(+) domain of the flagellar regulatory protein FlrC (FlrC(C)), a bEBP that controls flagellar synthesis in Vibrio cholerae. Our results showed that FlrC(C) forms heptamer both in nucleotide (Nt)-free and -bound states without ATP-dependent subunit remodeling. Unlike the bEBPs such as NtrC1 or PspF, a novel cis-mediated "all or none" ATP binding occurs in the heptameric FlrC(C), because constriction at the ATPase site, caused by loop L3 and helix α7, restricts the proximity of the trans-protomer required for Nt binding. A unique "closed to open" movement of Walker A, assisted by trans-acting "Glu switch" Glu-286, facilitates ATP binding and hydrolysis. Fluorescence quenching and ATPase assays on FlrC(C) and mutants revealed that although Arg-349 of sensor II, positioned by trans-acting Glu-286 and Tyr-290, acts as a key residue to bind and hydrolyze ATP, Arg-319 of α7 anchors ribose and controls the rate of ATP hydrolysis by retarding the expulsion of ADP. Heptameric state of FlrC(C) is restored in solution even with the transition state mimicking ADP·AlF3. Structural results and pulldown assays indicated that L3 renders an in-built geometry to L1 and L2 causing σ(54)-FlrC(C) interaction independent of Nt binding. Collectively, our results underscore a novel mechanism of ATP binding and σ(54) interaction that strives to understand the transcriptional mechanism of the bEBPs, which probably interact directly with the RNA polymerase-σ(54) complex without DNA looping.

Conformational barrier of CheY3 and inability of CheY4 to bind FliM control the flagellar motor action in Vibrio cholerae.

Vibrio cholerae contains multiple copies of chemotaxis response regulator (VcCheY1-VcCheY4) whose functions are elusive yet. Although previous studies suggested that only VcCheY3 directly switches the flagellar rotation, the involvement of VcCheY4 in chemotaxis could not be ruled out. None of these studies, however, focused on the structure, mechanism of activation or molecular basis of FliM binding of the VcCheYs. From the crystal structures of Ca(2+) and Mg(2+) bound VcCheY3 we proposed the presence of a conformational barrier composed of the hydrophobic packing of W61, M88 and V106 and a unique hydrogen bond between T90 and Q97 in VcCheY3. Lesser fluorescence quenching and higher Km value of VcCheY3, compared to its mutants VcCheY3-Q97A and VcCheY3-Q97A/E100A supported our proposition. Furthermore, aforesaid biochemical data, in conjunction with the structure of VcCheY3-Q97A, indicated that the coupling of T90 and Q97 restricts the movement of T90 toward the active site reducing the stabilization of the bound phosphate and effectively promoting autodephosphorylation of VcCheY3. The structure of BeF3(-) activated VcCheY3 insisted us to argue that elevated temperature and/or adequacy of phosphate pool might break the barrier of the free-state VcCheY3 and the conformational changes, required for FliM binding, occur upon phosphorylation. Structure of VcCheY4 has been solved in the free and sulfated states. VcCheY4(sulf), containing a bound sulfate at the active site, appears to be more compact and stable with a longer α4 helix, shorter ß4α4 loop and hydrogen bond between T82 and the sulfate compared to VcCheY4(free). While pull down assay of VcCheYs with VcFliMNM showed that only activated VcCheY3 can interact with VcFliMNM and VcCheY4 cannot, a knowledge based docking explained the molecular mechanism of the interactions between VcCheY3 and VcFliM and identified the limitations of VcCheY4 to interact with VcFliM even in its phosphorylated state.

Overexpression, purification, crystallization and preliminary X-ray analysis of CheY4 from Vibrio cholerae O395.

Chemotaxis and motility greatly influence the infectivity of Vibrio cholerae, although the role of chemotaxis genes in V. cholerae pathogenesis is poorly understood. In contrast to the single copy of CheY found in Escherichia coli and Salmonella typhimurium, four CheYs (CheY1-CheY4) are present in V. cholerae. While insertional disruption of the cheY4 gene results in decreased motility, insertional duplication of this gene increases motility and causes enhanced expression of the two major virulence genes. Additionally, cheY3/cheY4 influences the activation of the transcription factor NF-κB, which triggers the generation of acute inflammatory responses. V. cholerae CheY4 was cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY4 grown in space group C2 diffracted to 1.67 Å resolution, with unit-cell parameters a = 94.4, b = 31.9, c = 32.6 Å, ß = 96.5°, whereas crystals grown in space group P3(2)21 diffracted to 1.9 Å resolution, with unit-cell parameters a = b = 56.104, c = 72.283 Å, γ = 120°.

Cloning, overexpression, purification, crystallization and preliminary X-ray analysis of CheY3, a response regulator that directly interacts with the flagellar 'switch complex' in Vibrio cholerae.

Vibrio cholerae is the aetiological agent of the severe diarrhoeal disease cholera. This highly motile organism uses the processes of motility and chemotaxis to travel and colonize the intestinal epithelium. Chemotaxis in V. cholerae is far more complex than that in Escherichia coli or Salmonella typhimurium, with multiple paralogues of various chemotaxis genes. In contrast to the single copy of the chemotaxis response-regulator protein CheY in E. coli, V. cholerae contains four CheYs (CheY1-CheY4), of which CheY3 is primarily responsible for interacting with the flagellar motor protein FliM, which is one of the major constituents of the ;switch complex' in the flagellar motor. This interaction is the key step that controls flagellar rotation in response to environmental stimuli. CheY3 has been cloned, overexpressed and purified by Ni-NTA affinity chromatography followed by gel filtration. Crystals of CheY3 were grown in space group R3, with a calculated Matthews coefficient of 2.33 A3 Da(-1) (47% solvent content) assuming the presence of one molecule per asymmetric unit.