A family of fibrinogen-binding MSCRAMMs from Enterococcus faecalis.

We report that three (EF0089, EF2505 and EF1896, renamed here Fss1, Fss2 and Fss3, respectively, for Enterococcus faecalis surface protein) of the recently predicted MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) in E. faecalis strain V583 bind fibrinogen (Fg). Despite an absence of extensive primary sequence homology, the three proteins appear to be related structurally. Within the N-terminal regions of the three enterococcal proteins, we identified pairs of putative IgG-like modules with a high degree of predicted structural similarity to the Fg-binding N2 and N3 domains of the staphylococcal MSCRAMMs ClfA and SdrG. A second N2N3-like segment was predicted in Fss1. Far-UV circular dichroism spectroscopy revealed that all four predicted N2N3-like regions are composed mainly of beta-sheets with only a minor proportion of alpha-helices, which is characteristic of Ig-like folded domains. Three of the four identified enterococcal N2N3-like regions showed potent dose-dependent binding to Fg. However, the specificity of the Fg-binding MSCRAMMs differs, as indicated by far-Western blots, which showed that recombinant segments of the MSCRAMMs bound different Fg polypeptide chains. Enterococci grown in serum-supplemented broth adhere to Fg-coated surfaces, and inactivation in strain OG1RF of the gene encoding Fss2 resulted in reduced adherence, whilst complementation of the mutant restored full Fg adherence. Thus, E. faecalis contains a family of MSCRAMMs that structurally and functionally resemble the Fg-binding MSCRAMMs of staphylococci.

A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics.

The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) gamma-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin alpha(IIb)beta(3) and ClfA bind to the same segment in the Fg gamma-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the "Dock, Lock and Latch" mechanism previously described for the Staphylococcus epidermidis SdrG-Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not alpha(IIb)beta(3) may allow the design of novel anti-staphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may have originated from a common ancestor but have evolved to accommodate specific ligand structures.

Characterization of the West Nile virus protease substrate specificity and inhibitors.

West Nile virus (WNV), a mosquito-borne member of Flaviviridae, is a human pathogen causing widespread disease for which there is no vaccine or chemotherapy. The two-component viral serine protease consists of a heterodimeric complex between the hydrophilic domain of the cofactor, NS2B (NS2BH) and the protease domain (NS3-pro). The protease is essential for polyprotein processing followed by assembly of viral replicase and genome replication. Therefore, the protease is an excellent target for development of antiviral therapeutics. Here, we report the expression in Escherichia coli, purification, and characterization of biochemical and kinetic properties of the WNV protease. Furthermore, we show that the WNV and the dengue virus type 2 (DENV-2) proteases are inhibited by aprotinin with inhibitor constants of 0.16 and 0.026 microM, respectively. Molecular modeling of the WNV protease/aprotinin complex, based on the known crystal structures of the WNV NS2BH-N3pro and aprotinin, suggest a potentially strong interaction between the P2 Lys and the protease activator peptide, NS2BH. This conclusion based on molecular modeling is in agreement with our data of a higher k(cat)/Km value with the substrate, Boc-Gly-Lys-Arg-MCA than the Boc-Gly-Arg-Arg-MCA and is also consistent with the results of an earlier study that were based on substrate-based inhibitor peptides.

Structural basis for antagonism by suramin of heparin binding to vaccinia complement protein.

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.

Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities.

A series of 46 charged-to-alanine mutations in the yellow fever virus NS2B-NS3 protease, previously characterized in cell-free and transient cellular expression systems, was tested for their effects on virus recovery. Four distinct plaque phenotypes were observed in cell culture: parental plaque-size (13 mutants), reduced plaque-size (17 mutants), small plaque-size (8 mutants) and no plaque-formation (8 mutants). No mutants displayed any temperature sensitivity based on recovery of virus after RNA transfection at 32 versus 37 degrees C. Most small plaque-mutants were defective in growth efficiency compared with parental virus. However not all small plaque-mutants had defective 2B/3 cleavage, with some showing selective defects at other non-structural protein cleavage sites. Revertant viruses were recovered for six mutations that caused reduced plaque sizes. Same-site and second-site mutations occurred in NS2B, and one second-site mutation occurred in the NS3 protease domain. Some reversion mutations ameliorated defects in cleavage activity and plaque size caused by the original mutation. These data indicate that certain mutations that reduce NS2B-NS3 protease cleavage activity cause growth restriction of yellow fever virus in cell culture. However, for at least two mutations, processing defects other than impaired cleavage activity at the 2B/3 site may account for the mutant phenotype. The existence of reversion mutations primarily in NS2B rather than NS3, suggests that the protease domain is less tolerant of structural perturbation compared with the NS2B protein.

Identification and characterization of nonsubstrate based inhibitors of the essential dengue and West Nile virus proteases.

The 72 known members of the flavivirus genus include lethal human pathogens such as Yellow Fever, West Nile, and Dengue viruses. There is at present no known chemotherapy for any flavivirus and no effective vaccines for most. A common genomic organization and molecular mechanisms of replication in hosts are shared by flaviviruses with a viral serine protease playing a pivotal role in processing the viral polyprotein into component polypeptides, an obligatory step in viral replication. Using the structure of the dengue serine protease complexed with a protein inhibitor as a template, we have identified five compounds, which inhibit the enzyme. We also describe parallel inhibitory activity of these compounds against the West Nile virus Protease. A few of the compounds appear to provide a template for design of more potent and specific inhibitors of the dengue and West Nile virus proteases. Sequence similarities among flaviviral proteases suggests that such compounds might also possibly inhibit other flaviviral proteases.