Crystal structure of type I 3-dehydroquinate dehydratase of Aquifex aeolicus suggests closing of active site flap is not essential for enzyme action.

Structural analyses of enzymes involved in biosynthetic pathways that are present in micro-organisms, but absent from mammals (for example Shikimate pathway) are important in developing anti-microbial drugs. Crystal structure of the Shikimate pathway enzyme, type I 3-dehydroquinate dehydratase (3-DHQase) from the hyperthermophilic bacterium Aquifex aeolicus was solved both as an apo form and in complex with a ligand. The complex structure revealed an interesting structural difference when compared to other ligand-bound type I 3-DHQases suggesting that closure of the active site loop is not essential for catalysis. This provides new insights into the catalytic mechanism of type I 3-DHQases.

Collagen adhesin-nanoparticle interaction impairs adhesin's ligand binding mechanism.

BACKGROUND: Pathogenic bacteria specifically recognize extracellular matrix (ECM) molecules of the host (e.g. collagen, fibrinogen and fibronectin) through their surface proteins known as MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) and initiate colonization. On implantation, biomaterials easily get coated with these ECM molecules and the MSCRAMMs mediate bacterial adherence to biomaterials. With the rapid rise in antibiotic resistance, designing alternative strategies to reduce/eliminate bacterial colonization is absolutely essential. METHODS: The Rhusiopathiae surface protein B (RspB) is a collagen-binding MSCRAMM of Erysipelothrix rhusiopathiae. It also binds to abiotic surfaces. The crystal structure of the collagen-binding region of RspB (rRspB31-348) reported here revealed that RspB also binds collagen by a unique ligand binding mechanism called "Collagen Hug" which is a common theme for collagen-binding MSCRAMMs of many Gram-positive bacteria. Here, we report the interaction studies between rRspB31-348 and silver nanoparticles using methods like gel shift assay, gel permeation chromatography and circular dichroism spectroscopy. RESULTS: The "Collagen Hug" mechanism was inhibited in the presence of silver nanoparticles as rRspB31-348 was unable to bind to collagen. The total loss of binding was likely because of rRspB31-348 and silver nanoparticle protein corona formation and not due to the loss of the structural integrity of rRspB31-348 on binding with nanoparticles as observed from circular dichroism experiments. GENERAL SIGNIFICANCE: Interaction of rRspB31-348 with silver nanoparticle impaired its ligand binding mechanism. Details of this inhibition mechanism may be useful for the development of antimicrobial materials and antiadhesion drugs.

Cloning, expression, purification and ligand binding studies of novel fibrinogen-binding protein FbsB of Streptococcus agalactiae.

Fibrinogen (Fg) is often a common site for bacterial recognition. In Streptococcus agalactiae, two surface proteins that recognize Fg are FbsA and FbsB. FbsA and the N-terminal region of FbsB have been shown to bind to human Fg, while the C-terminal region of FbsB [FbsB(C)] has been speculated to bind to bovine Fg. This C-terminal region which is conserved in many of the S. agalactiae strains was tested for binding to bovine Fg. For this, FbsB(C) was cloned, expressed and purified. Dot blot, Western blot and ELISA experiments carried out with the purified protein showed that FbsB(C) has the ability to bind to bovine Fg. It was also observed that other than binding to the native form of Fg, FbsB(C) also has the ability to bind to the Fg subunits when reduced. On studying the influence of Ca(2+) on the FbsB(C)-bovine Fg binding it was observed that the addition of Ca(2+) in the assay experiment greatly stimulated the binding. When the primary structure of FbsB(C) was analyzed, it was seen that other than similarities with strains of the same organism, it does not have any similarity with any protein characterized so far. In addition to this, its secondary structure component analysis by circular dichroism revealed that it is composed mainly of alpha helices and random coils unlike other Fg-binding surface proteins where beta sheets are dominant. FbsB(C) indeed is a novel protein and understanding the mechanism of its interaction with Fg would be useful in developing strategies to fight against infections by Streptococcus.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the collagen-binding region of RspB from Erysipelothrix rhusiopathiae.

RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB((31-348)), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5 A using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2 A using synchrotron radiation. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 46.19, b = 66.65, c = 101.72 A, beta = 94.11 degrees .