Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems.

RNA interference by type III CRISPR systems results in the synthesis of cyclic oligoadenylate (cOA) second messengers, which are known to bind and regulate various CARF domain-containing nuclease receptors. The CARF domain-containing Csa3 family of transcriptional factors associated with the DNA-targeting type I CRISPR systems regulate expression of various CRISPR and DNA repair genes in many prokaryotes. In this study, we extend the known receptor repertoire of cOA messengers to include transcriptional factors by demonstrating specific binding of cyclic tetra-adenylate (cA4) to Saccharolobus solfataricus Csa3 (Csa3Sso). Our 2.0-Å resolution X-ray crystal structure of cA4-bound full-length Csa3Sso reveals the binding of its CARF domain to an elongated conformation of cA4. Using cA4 binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface, we identified a Csa3-specific cA4 binding motif distinct from a more widely conserved cOA-binding CARF motif. Using a rational surface engineering approach, we increased the cA4 binding affinity of Csa3Sso up to ∼145-fold over the wildtype, which has potential applications for future second messenger-driven CRISPR gene expression and editing systems. Our in-solution Csa3Sso structural analysis identified cA4-induced allosteric and asymmetric conformational rearrangement of its C-terminal winged helix-turn-helix effector domains, which could potentially be incompatible to DNA binding. However, specific in vitro binding of the purified Csa3Sso to its putative promoter (PCas4a) was found to be cA4 independent, suggesting a complex mode of Csa3Sso regulation. Overall, our results support cA4-and Csa3-mediated cross talk between type III and type I CRISPR systems.

Structural basis of KdpD histidine kinase binding to the second messenger c-di-AMP.

The KdpDE two-component system regulates potassium homeostasis and virulence in various bacterial species. The KdpD histidine kinases (HK) of this system contain a universal stress protein (USP) domain which binds to the second messenger cyclic-di-adenosine monophosphate (c-di-AMP) for regulating transcriptional output from this two-component system in Firmicutes such as Staphylococcus aureus. However, the structural basis of c-di-AMP specificity within the KdpD-USP domain is not well understood. Here, we resolved a 2.3 Å crystal structure of the S. aureus KdpD-USP domain (USPSa) complexed with c-di-AMP. Binding affinity analyses of USPSa mutants targeting the observed USPSa:c-di-AMP structural interface enabled the identification of the sequence residues that are required for c-di-AMP specificity. Based on the conservation of these residues in other Firmicutes, we identified the binding motif, (A/G/C)XSXSX2N(Y/F), which allowed us to predict c-di-AMP binding in other KdpD HKs. Furthermore, we found that the USPSa domain contains structural features distinct from the canonical standalone USPs that bind ATP as a preferred ligand. These features include inward-facing conformations of its ß1-α1 and ß4-α4 loops, a short α2 helix, the absence of a triphosphate-binding Walker A motif, and a unique dual phospho-ligand binding mode. It is therefore likely that USPSa-like domains in KdpD HKs represent a novel subfamily of the USPs.

Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species.

Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCEVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.

Probing the inhibitory potency of epigallocatechin gallate against human γB-crystallin aggregation: Spectroscopic, microscopic and simulation studies.

Aggregation of human ocular lens proteins, the crystallins is believed to be one of the key reasons for age-onset cataract. Previous studies have shown that human γD-crystallin forms amyloid like fibres under conditions of low pH and elevated temperature. In this article, we have investigated the aggregation propensity of human γB-crystallin in absence and presence of epigallocatechin gallate (EGCG), in vitro, when exposed to stressful conditions. We have used different spectroscopic and microscopic techniques to elucidate the inhibitory effect of EGCG towards aggregation. The experimental results have been substantiated by molecular dynamics simulation studies. We have shown that EGCG possesses inhibitory potency against the aggregation of human γB-crystallin at low pH and elevated temperature.

Macroscopic amyloid fiber formation by staphylococcal biofilm associated SuhB protein.

Staphylococcus aureus is a commensal and opportunistic pathogen that causes lethal infections. Biofilm forming ability of S. aureus enhances its virulence since biofilm provides the bacteria protective shield against antibiotics and host immunity. Polysaccharide independent biofilm formation by several virulent S. aureus strains have been identified recently, where protein components substitute polysaccharide intercellular adhesin (PIA) involved in bacterial cell attachment. The suhB gene has been reported to be essential in staphylococcal PIA-independent biofilm formation. Overexpression of staphylococcal SuhB (SasuhB) in E. coli produces extracellular macroscopic fibers made of recombinant SaSuhB protein. The amyloidic nature of the fiber is evaluated by high resolution electron microscopy, X-ray fiber diffraction and amyloid specific dyes, such as Congo red and thioflavin-T binding assay. The fibers appear to be sticky in nature and bind a large number of bacterial cells. The results suggest the possible role of SaSuhB-fibers as a structural component as well as an adhesin in biofilm matrix.

Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8- and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel ß strands stacked between α helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely (127)ALDITLPMPPR(137) and (138)WTQVPDPNVPDAFVVIADR(156),are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping.

Structural elucidation of the NADP(H) phosphatase activity of staphylococcal dual-specific IMPase/NADP(H) phosphatase.

NADP(H)/NAD(H) homeostasis has long been identified to play a pivotal role in the mitigation of reactive oxygen stress (ROS) in the intracellular milieu and is therefore critical for the progression and pathogenesis of many diseases. NAD(H) kinases and NADP(H) phosphatases are two key players in this pathway. Despite structural evidence demonstrating the existence and mode of action of NAD(H) kinases, the specific annotation and the mode of action of NADP(H) phosphatases remains obscure. Here, structural evidence supporting the alternative role of inositol monophosphatase (IMPase) as an NADP(H) phosphatase is reported. Crystal structures of staphylococcal dual-specific IMPase/NADP(H) phosphatase (SaIMPase-I) in complex with the substrates D-myo-inositol-1-phosphate and NADP(+) have been solved. The structure of the SaIMPase-I-Ca(2+)-NADP(+) ternary complex reveals the catalytic mode of action of NADP(H) phosphatase. Moreover, structures of SaIMPase-I-Ca(2+)-substrate complexes have reinforced the earlier proposal that the length of the active-site-distant helix α4 and its preceding loop are the predisposing factors for the promiscuous substrate specificity of SaIMPase-I. Altogether, the evidence presented suggests that IMPase-family enzymes with a shorter α4 helix could be potential candidates for previously unreported NADP(H) phosphatase activity.

Functional insights from molecular modeling, docking, and dynamics study of a cypoviral RNA dependent RNA polymerase.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) contains 11 double stranded RNA genome segments and infects tasar silkworm A. mylitta. RNA-dependent RNA polymerase (RdRp) is reported as a key enzyme responsible for propagation of the virus in the host cell but its structure function relationship still remains elusive. Here a computational approach has been taken to compare sequence and secondary structure of AmCPV RdRp with other viral RdRps to identify consensus motifs. Then a reliable pairwise sequence alignment of AmCPV RdRp with its closest sequence structure homologue λ3 RdRp is done to predict three dimensional structure of AmCPV RdRp. After comparing with other structurally known viral RdRps, important sequence and/or structural features involved in substrate entry or binding, polymerase reaction and the product release events have been identified. A conserved RNA pentanucleotide (5'-AGAGC-3') at the 3'-end of virus genome is predicted as cis-acting signal for RNA synthesis and its docking and simulation study along with the model of AmCPV RdRp has allowed to predict mode of template binding by the viral polymerase. It is found that template RNA enters into the catalytic center through nine sequence-independent and two sequence-dependent interactions with the specific amino acid residues. However, number of sequence dependent interactions remains almost same during 10 nano-second simulation time while total number of interactions decreases. Further, docking of N(7)-methyl-GpppG (mRNA cap) on the model as well as prediction of RNA secondary structure has shown the template entry process in the active site. These findings have led to postulate the mechanism of RNA-dependent RNA polymerization process by AmCPV RdRp. To our knowledge, this is the first report to evaluate structure function relationship of a cypoviral RdRp.

Crystal structure of dehydratase component HadAB complex of mycobacterial FAS-II pathway.

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like ß-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.

Structural elucidation of the binding site and mode of inhibition of Li(+) and Mg(2+) in inositol monophosphatase.

UNLABELLED: Mg(2+) -dependent, Li(+) -sensitive phosphatases are a widely distributed family of enzymes with significant importance throughout the biological kingdom. Inositol monophosphatase (IMPase) is an important target of Li(+) -based therapeutic agents in manic depressive disorders. However, despite decades of intense research efforts, the precise mechanism of Li(+) -induced inhibition of IMPase remains obscured. Here we describe a structural investigation of the Li(+) binding site in staphylococcal IMPase I (SaIMPase I) using X-ray crystallography. The biochemical study indicated common or overlapping binding sites for Mg(2+) and Li(+) in the active site of SaIMPase I. The crystal structure of the SaIMPase I ternary product complex shows the presence of a phosphate and three Mg(2+) ions (namely Mg1, Mg2 and Mg3) in the active site. As Li(+) is virtually invisible in X-ray crystallography, competitive displacement of Mg(2+) ions from the SaIMPase I ternary product complex as a function of increasing LiCl concentration was used to identify the Li(+) binding site. In this approach, the disappearing electron density of Mg(2+) ions due to Li(+) ion binding was traced, and the Mg(2+) ion present at the Mg2 binding site was found to be replaced. Moreover, based on a detailed comparative investigation of the phosphate orientation and coordination states of Mg(2+) binding sites in enzyme-substrate and enzyme-product complexes, inhibition mechanisms for Li(+) and Mg(2+) are proposed. DATABASE: The atomic coordinates for the SaIMPase I ternary complex, SaIMPase I in 50 mm LiCl, SaIMPase I in 100 mm LiCl and SaIMPase I in 0 mm MgCl2 have been submitted to the Protein Data Bank under accession numbers 4G61, 4I40, 4I3Y and 4PTK, respectively.

Cloning, expression, crystallization and preliminary X-ray diffraction studies of staphylococcal superantigen-like protein 1 (SSL1).

Staphylococcus aureus produces a family of exotoxins which are structural homologues of superantigens and thus are called staphylococcal superantigen-like proteins (SSLs). Amongst the 14 SSL genes, ssl1 (SAOUHSC_00383) has been cloned in the pQE30 expression vector, overexpressed in Escherichia coli M15 (pREP4) cells and the protein purified to homogeneity. The protein was crystallized using 6% Tacsimate pH 6.0, 0.1 M MES pH 6.0, 25%(w/v) polyethylene glycol 3350, 100 mM NDSB 256 at 298 K by the sitting-drop vapour-diffusion method. The crystals belonged to space group P21, with unit-cell parameters a = 77.9, b = 70.5, c = 126.5 Å, ß = 106.2°. X-ray diffraction data were collected and processed to a maximum resolution of 2.5 Å. The crystal contains six molecules in the asymmetric unit.