Novel fold and assembly of the repetitive B region of the Staphylococcus aureus collagen-binding surface protein.

BACKGROUND: [corrected] The Staphylococcus aureus collagen-binding protein Cna mediates bacterial adherence to collagen. The primary sequence of Cna has a non-repetitive collagen-binding A region, followed by the repetitive B region. The B region has one to four 23 kDa repeat units (B(1)-B(4)), depending on the strain of origin. The affinity of the A region for collagen is independent of the B region. However, the B repeat units have been suggested to serve as a 'stalk' that projects the A region from the bacterial surface and thus facilitate bacterial adherence to collagen. To understand the biological role of these B-region repeats we determined their three-dimensional structure. RESULTS: B(1) has two domains (D(1) and D(2)) placed side-by-side. D(1) and D(2) have similar secondary structure and exhibit a unique fold that resembles but is the inverse of the immunoglobulin-like (IgG-like) domains. Comparison with similar immunoglobulin superfamily (IgSF) structures shows novel packing arrangements between the D(1) and D(2) domains. In the B(1)B(2) crystal structure, an omission of a single glycine residue in the D(2)-D(3) linker loop, compared to the D(1)-D(2) and D(3)-D(4) linker loops, resulted in projection of the D(3) and D(4) in a spatially new orientation. We also present a model for B(1)B(2)B(3)B(4). CONCLUSIONS: The B region of the Cna collagen adhesin has a novel fold that is reminiscent of but is inverse in nature to the IgG fold. This B region assembly could effectively provide the needed flexibility and stability for presenting the ligand binding A region away from the bacterial cell surface.

Advances in surface plasmon resonance biosensor analysis.

The number and diversity of surface plasmon resonance (SPR) biosensor applications continue to increase. Evolutions in instrument and sensor chip technology, experimental methodology, and data analysis are making it possible to examine a wider variety of biomolecular interactions in greater mechanistic detail. SPR biosensors are poised to make a significant impact in basic research and pharmaceutical discovery.

Survey of the 1999 surface plasmon resonance biosensor literature.

The application of surface plasmon resonance biosensors in life sciences and pharmaceutical research continues to increase. This review provides a comprehensive list of the commercial 1999 SPR biosensor literature and highlights emerging applications that are of general interest to users of the technology. Given the variability in the quality of published biosensor data, we present some general guidelines to help increase confidence in the results reported from biosensor analyses.

Survey of the year 2000 commercial optical biosensor literature.

We have compiled a comprehensive list of the articles published in the year 2000 that describe work employing commercial optical biosensors. Selected reviews of interest for the general biosensor user are highlighted. Emerging applications in areas of drug discovery, clinical support, food and environment monitoring, and cell membrane biology are emphasized. In addition, the experimental design and data processing steps necessary to achieve high-quality biosensor data are described and examples of well-performed kinetic analysis are provided.

BIACORE J: a new platform for routine biomolecular interaction analysis.

SPR biosensor technology continues to evolve. The recently released platform from Biacore AB (Uppsala, Sweden), BIACORE J, is designed for the routine analysis of biomolecular interactions. Using an antibody-protein A and a ligand-receptor system, we demonstrate the utility of BIACORE J in determining active concentration and binding affinities. The results from these studies illustrate the high sensitivity of the instrument and its ability to generate reproducible binding responses. The BIACORE J is easy to operate and useful in diverse applications, making SPR technology widely accessible as a research tool.

Synthesis and spectral characterization of 5'-phosphopyridoxyl-D,L-7-azatryptophan, a photophysical probe of protein structure and dynamics.

We report the first isolation of a unique adduct of pyridoxal 5'-phosphate, 5'-phosphopyridoxyl-D,L-7-azatryptophan, and suggest a new and easier route for synthesis and purification of 5'-phosphopyridoxyl-L-(or -D-)tryptophan. The absorbance and emission spectra of the 7-azatryptophan adduct are distinctly different than those of pyridoxal 5'-phosphate or the tryptophan adduct. We propose that 5'-phosphopyridoxyl-D,L-7-azatryptophan has great potential as an intrinsic probe in optical studies of protein dynamics.

Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex.

Apoptosis is a highly regulated process that is crucial for normal development and homeostasis of multicellular organisms. The p35 protein from baculoviruses effectively prevents apoptosis by its broad-spectrum caspase inhibition. Here we report the crystal structure of p35 in complex with human caspase-8 at 3.0 A resolution, and biochemical and mutagenesis studies based on the structural information. The structure reveals that the caspase is inhibited in the active site through a covalent thioester linkage to p35, which we confirmed by gel electrophoresis, hydroxylamine treatment and mass spectrometry experiments. The p35 protein undergoes dramatic conformational changes on cleavage by the caspase. The repositioning of the amino terminus of p35 into the active site of the caspase eliminates solvent accessibility of the catalytic dyad. This may be crucial for preventing hydrolysis of the thioester intermediate, which is supported by the abrogation of inhibitory activity through mutations at the N terminus of p35. The p35 protein also makes conserved contacts with the caspase outside the active-site region, providing the molecular basis for the broad-spectrum inhibitory activity of this protein. We demonstrate a new molecular mechanism of caspase inhibition, as well as protease inhibition in general.

High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE.

Characterizing how chemical compounds bind to human serum albumin (HSA) is essential in evaluating drug candidates. Using warfarin as a test system, we validate the application of BIACORE SPR biosensors to reliably determine binding constants for drug/HSA interactions. The binding responses for warfarin over HSA surfaces were extremely reproducible even though warfarin is small compared to the size of the immobilized protein. At high concentrations, warfarin bound at more than one site on HSA, which is consistent with its known binding properties. The affinity we determined for the high-affinity site (K(25 degrees C)(d) = 3.7 +/- 1.2 microM), as well as the dissociation rate constant (k(25 degrees C)(d) = 1.2 s(-1)), are also consistent with binding constants determined previously. These results validate the biosensor technology and illustrate how BIACORE can be used to study drug/HSA interactions in a high-resolution mode. Using a set of 10 test compounds, we present a protocol for determining equilibrium dissociation constants for HSA in a high-throughput mode. Our method involves working at low compound concentrations and fitting the equilibrium data for all compounds simultaneously. We show that the % bound values determined by SPR correlate with the values determined by solution-based methods. The ability to examine directly the binding of small molecules (130-800 Da), coupled with minimal sample requirements and automated instrumentation, makes BIACORE technology applicable for evaluating drug/HSA interactions.

Structural basis of caspase inhibition by XIAP: differential roles of the linker versus the BIR domain.

The inhibitor of apoptosis proteins (IAPs) represent the only endogenous caspase inhibitors and are characterized by the presence of baculoviral IAP repeats (BIRs). Here, we report the crystal structure of the complex between human caspase-7 and XIAP (BIR2 and the proceeding linker). The structure surprisingly reveals that the linker is the only contacting element for the caspase, while the BIR2 domain is invisible in the crystal. The linker interacts with and blocks the substrate groove of the caspase in a backward fashion, distinct from substrate recognition. Structural analyses suggest that the linker is the energetic and specificity determinant of the interaction. Further biochemical characterizations clearly establish that the linker harbors the major energetic determinant, while the BIR2 domain serves as a regulatory element for caspase binding and Smac neutralization.

Biochemical analyses of the interactions between human immunodeficiency virus type 1 Vpr and p6(Gag).

The nonstructural human immunodeficiency virus type 1 Vpr protein is packaged into progeny virions at significant levels (approximately 200 copies/virion). Genetic analyses have demonstrated that efficient Vpr packaging is dependent upon a leucine-X-X-leucine-phenylalanine (LXXLF) motif located in the p6(Gag) domain of the structural Gag polyprotein. Recombinant proteins spanning full-length Vpr (Vpr(1-97)) or the amino-terminal 71 amino acids (Vpr(1-71)) formed specific complexes with recombinant p6 proteins in vitro. Complex formation required an intact LXXLF motif and exhibited an intrinsic dissociation constant of approximately 75 microM. Gel filtration and cross-linking analyses further revealed that Vpr(1-71) self-associated in solution. Our experiments demonstrate that Vpr can bind directly and specifically to p6 and suggest that oligomerization of both Vpr and Gag may serve to increase the avidity and longevity of Vpr-Gag complexes, thereby ensuring efficient Vpr packaging.

A novel mechanism of TRAF signaling revealed by structural and functional analyses of the TRADD-TRAF2 interaction.

TRAF proteins are major mediators for the cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily. They can be recruited to activated TNF receptors either by direct interactions with the receptors or indirectly via the adaptor protein TRADD. We now report the structure of the TRADD-TRAF2 complex, which is highly distinct from receptor-TRAF2 interactions. This interaction is significantly stronger and we show by an in vivo signaling assay that TRAF2 signaling is more readily initiated by TRADD than by direct receptor-TRAF2 interactions. TRADD is specific for TRAF1 and TRAF2, which ensures the recruitment of clAPs for the direct inhibition of caspase activation in the signaling complex. The stronger affinity and unique specificity of the TRADD-TRAF2 interaction are crucial for the suppression of apoptosis and provide a mechanistic basis for the perturbation of TRAF recruitment in sensitizing cell death induction.

Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1.

Integrins alpha(1)beta(1) and alpha(2)beta(1) are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the alpha chains. Previously, we reported that recombinant A-domain of alpha(1)beta(1) (alpha(1)A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999) J. Biol. Chem. 274, 24906-24913). Here, we compared the binding of the recombinant A-domain of alpha(2)beta(1) (alpha(2)A) to type I collagen with that of alpha(1)A using surface plasmon resonance and showed that alpha(2)A exhibited only one detectable class of binding sites in type I collagen, with a K(D) of approximately 10 microm at approximately 3 binding sites per collagen molecule. We further demonstrated that alpha(1)A and alpha(2)A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of alpha(1)A- and alpha(2)A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for alpha(1)A and alpha(2)A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both alpha(1)A and alpha(2)A (Knight, C. G., et al. (2000) J. Biol. Chem. 275, 35-40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of alpha(1)A and alpha(2)A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for alpha(1)A and alpha(2)A.

Domain structure of the Staphylococcus aureus collagen adhesin.

Sequence analysis of surface proteins from Gram-positive bacteria indicates a composite organization consisting of unique and repeated segments. Thus, these proteins may contain discrete domains that could fold independently. In this paper, we have used a panel of biophysical methods, including gel permeation chromatography, analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy, to analyze the structural organization of the Staphylococcus aureus collagen adhesin, CNA. Our results indicate that the structure, function, and folding of the ligand-binding domain (A) are not affected by the presence or absence of the other major domain (B). In addition, little or no interaction is observed between the nearly identical repeat units within the B domain. We propose that CNA is indeed a mosaic protein in which the different domains previously indicated by sequence analysis operate independently.

Ace is a collagen-binding MSCRAMM from Enterococcus faecalis.

A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174-319) suggested a structure very similar to that reported for residues 151-318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a beta-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833-838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.

Crystallization and preliminary X-ray analysis of B-domain fragments of a Staphylococcus aureus collagen-binding protein.

Recombinant proteins of monomeric and dimeric B-domain repeats of a Staphylococcus aureus FDA 574 collagen-binding adhesin have been crystallized. The single repeat unit (B1) was crystallized in a body-centered orthorhombic lattice with a = 96.9, b = 101.3, c = 120. 8 A in either the I222 or I212121 space group. These crystals diffracted to 2.5 A resolution and the calculated Vm values of 3.2 and 2.2 A3 Da-1 suggest the possibility of a dimer or a trimer in the asymmetric unit. The two-repeat fragment (B1B2) crystallized in the orthorhombic space group P212121 with cell dimensions a = 42.4, b = 79.4, c = 130.4 A and diffracted to 2.3 A resolution. For this species, the calculated Vm value of 2.2 A3 Da-1 indicates the presence of a monomer in the asymmetric unit.

Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, alpha(1)beta(1) integrin and Staphylococcus aureus cna MSCRAMM.

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin alpha(2)beta(1) and a Staphylococcus aureus adhesin reveal a "trench" on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833-838). We report here the crystal structure of the alpha subunit I domain from the alpha(1)beta(1) integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human alpha(1) integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.