Evasion of Toll-like receptor 2 activation by staphylococcal superantigen-like protein 3.

Toll-like receptors (TLRs) are crucial for our host defense against microbial infections. TLR2 is especially important to fight bacterial infections, as it specifically recognizes bacterial lipoproteins of both Gram-positive and Gram-negative origin. Present on a variety of immune cells, TLR2 is critical for host protection against several bacterial infections, including those caused by Staphylococcus aureus. This major human pathogen causes increasing health care problems due to its increased resistance to antibiotics. S. aureus secretes a wide variety of proteins that inhibit innate immune responses. Recently, several staphylococcal superantigen-like proteins (SSLs) have been described to mediate immune evasive properties. Here, we describe that SSL3 specifically binds and inhibits TLR2 activation on human and murine neutrophils and monocytes. Through binding of the extracellular TLR2 domain, SSL3 inhibits IL-8 production by HEK cells expressing TLR1/2 and TLR2/6 dimers, stimulated with their specific ligands. The SSL3-TLR2 interaction is partially glycan dependent as binding of SSL3 to TLR2 is affected upon removal of sialic acid residues. Moreover, the SSL3(R308A) mutant lacking glycan-binding properties shows lower TLR2 inhibition. An SSL3 mutant, lacking the N-terminal 126 amino acids, still retains full TLR2 inhibiting activity. Of other SSLs tested, only SSL4, which shares the highest homology with SSL3, blocks TLR2 activation. SSL3 is the first-described bacterial protein that blocks TLR2 activation through direct extracellular interaction with the receptor. This unique function of SSL3 adds to the arsenal of immune evasive molecules that S. aureus can employ to subvert both innate and adaptive immunity.

Characterization of bitiscetin-2, a second form of bitiscetin from the venom of Bitis arietans : comparison of its binding site with the collagen-binding site on the von Willebrand factor A3-domain.

BACKGROUND: Bitiscetin, a heterodimeric snake venom protein purified from Bitis arietans, binds to the A1 domain of von Willebrand factor (VWF) and induces binding of this domain to platelet glycoprotein (GP) Ib. We previously purified a distinct form of dimeric bitiscetin (herein called bitiscetin-2) that also induces the VWF A1 domain-GPIb interaction, but does not bind to the A1 domain. Instead, it interacts with the collagen-binding A3 domain of VWF. METHODS: In the current study we identify the amino terminal sequence of the bitiscetin-2 as DEGCLPDDSSRT, showing conclusively that the protein is distinct form the originally described bitiscetin. We further studied the interaction of bitiscetin-2 and VWF using DeltaA3 VWF and a series of 33 VWF point mutants previously prepared to map the collagen-binding site. RESULTS: Our results confirm that DeltaA3 VWF, even though containing the A1-domain, is unable to interact with bitiscetin-2. Mutation of VWF-A3 residues Ile975, Asp979, Pro981, Ser1020 and His1023 reduces binding by 80% while mutation of residues Val980, Glu1001 and Arg1021 reduces binding by 30-60%. A 2- to 6-fold increase of binding is caused by mutation of residues Val985, Glu987, and Arg1016. CONCLUSION: Nearly all of these mutations also affect collagen binding showing that the binding sites for bitiscetin-2 and collagen type III in the VWF-A3 domain closely overlap.

Crystallization of quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni: crystals with unique optical properties.

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni is a functional electron-transfer protein containing both a haem c and a pyrroloquinoline quinone cofactor. The enzyme has been crystallized at 277 K using polyethylene glycol 6000 as precipitant. The crystals belong to space group C2, with unit-cell parameters a = 98.1, b = 74.3, c = 92.2 A, beta = 105.9 degrees. A native data set with a resolution of 2.44 A resolution has been collected. The approximate orientation of the haem group with respect to the unit-cell axes has been determined from the optical properties of the crystals.

Five novel mutations in the gene for human blood coagulation factor V associated with type I factor V deficiency.

Coagulation factor V (FV) plays an important role in maintaining the hemostatic balance in both the formation of thrombin in the procoagulant pathway as well as in the protein C anticoagulant pathway. FV deficiency is a rare bleeding disorder with variable phenotypic expression. Little is known about the molecular basis underlying this disease. This study identified 5 novel mutations associated with FV deficiency in 3 patients with severe FV deficiency but different clinical expression and 2 unaffected carriers. Four mutations led to a premature termination codon either by a nonsense mutation (single-letter amino acid codes): A1102T, K310Term. (FV Amersfoort) and C2491T, Q773Term. (FV Casablanca) or a frameshift: an 8-base pair deletion between nucleotides 1130 and 1139 (FV Seoul(1)) and a 1-base pair deletion between nucleotides 4291 and 4294 (FV Utrecht). One mutation was a novel missense mutation: T1927C, C585R (FV Nijkerk), resulting in the absence of mutant protein despite normal transcription to RNA. Most likely, an arginine at this position disrupts the hydrophobic interior of the FV A2 domain. The sixth detected mutation was a previously reported missense mutation: A5279G, Y1702C (FV Seoul(2)). In all cases, the presence of the mutation was associated with type I FV deficiency. Identifying the molecular basis of mutations underlying this rare coagulation disorder will help to obtain more insight into the mechanisms involved in the variable clinical phenotype of patients with FV deficiency.

Epitope mapping of inhibitory antibodies against platelet glycoprotein Ibalpha reveals interaction between the leucine-rich repeat N-terminal and C-terminal flanking domains of glycoprotein Ibalpha.

The interaction of von Willebrand factor (vWF) with the platelet receptor glycoprotein Ibalpha (GPIbalpha) is important for platelet adhesion at high shear stress. Two functionally important antigenic areas within GPIbalpha were identified through the characterization of 5 new inhibitory anti-GPIb monoclonal antibodies (mAbs). The binding sites of 3 of these anti-GPIb mAbs, which were intercompeting and potently inhibiting shear stress-induced binding of vWF, were mapped within the N-terminal amino acid (aa) 1-59 area by the use of canine-human chimeras. These antibodies, however, had little or no effect (approximately 40% inhibition) on the binding of vWF induced by either botrocetin or ristocetin. On the other hand, the anti-GPIb mAbs 24G10 and 6B4, which blocked GPIb-vWF binding under all conditions examined, bound to 2 different regions of GPIbalpha, aa 1-81 and aa 201-268, respectively. The epitope for 6B4 was further narrowed by phage display revealing 2 sets of peptide sequences aligning within aa 259-262 and aa 230-242. In the latter region of GPIbalpha, the gain-of-function platelet-type von Willebrand disease (PT-vWD) mutations have been identified. Alignment was partially confirmed because the binding of 6B4 to recombinant GPIbalpha fragments carrying either one of the PT-vWD mutations was considerably impaired but not completely abolished. In contrast, mAb 24G10 bound more strongly to mutant PT-vWD GPIbalpha. However, although 24G10 competed with 6B4 for binding to platelets, it bound to an epitope within aa 1-81 of GPIbalpha. In conclusion, 2 functionally important areas within GPIbalpha were identified: one localized within the leucine-rich repeat N-terminal aa 1-59 area and one composed of residues aa 1-81 in close contact with aa 201-268. Moreover, further support is provided for the existence of an intramolecular interaction between the N-terminal flanking (aa 1-81) and C-terminal flanking (aa 201-268) regions. (Blood. 2001;98:652-660)

The structure of leech anti-platelet protein, an inhibitor of haemostasis.

Leech anti-platelet protein (LAPP) from the leech Haementeria officinalis is a collagen-binding protein that inhibits the collagen-mediated adhesion of blood platelets. The crystal structure of recombinant LAPP has been determined using single isomorphous replacement with anomalous scattering combined with solvent flattening and threefold molecular averaging. The model of LAPP has been refined to 2.2 A resolution (R factor 21.5%; free R factor 24.0%). LAPP contains an 89-residue C-terminal domain consisting of a central six-stranded antiparallel beta-sheet flanked on one side by an alpha-helix and on the other side by two extended loops with little secondary structure. A 36-residue N-terminal region is not visible in the electron-density map. This region is rich in glycine and lacks hydrophobic residues. It probably does not have a compact globular fold, but instead has an extended conformation and is flexible. The crystal packing suggests that LAPP may form tightly interacting dimers. The fold of the C-terminal domain of LAPP closely resembles that of the N-domain of hepatocyte growth factor (HGF), which classifies LAPP as a PAN domain. However, no significant sequence homology exists between LAPP and other PAN domains. Common structural features between LAPP and the HGF N-domain include two disulfide bonds that link the alpha-helix to the central region of the protein and five residues with a conserved hydrophobic nature that are located in the core of the domain. These conserved structural features may be an important determinant of the PAN-domain type of fold.

Identification of the collagen-binding site of the von Willebrand factor A3-domain.

Von Willebrand factor (vWF) is a multimeric glycoprotein that mediates platelet adhesion and thrombus formation at sites of vascular injury. vWF functions as a molecular bridge between collagen and platelet receptor glycoprotein Ib. The major collagen-binding site of vWF is contained within the A3 domain, but its precise location is unknown. To localize the collagen-binding site, we determined the crystal structure of A3 in complex with an Fab fragment of antibody RU5 that inhibits collagen binding. The structure shows that RU5 recognizes a nonlinear epitope consisting of residues 962-966, 981-997, and 1022-1026. Alanine mutants were constructed of residues Arg(963), Glu(987), His(990), Arg(1016), and His(1023), located in or close to the epitope. Mutants were expressed as fully processed multimeric vWF. Mutation of His(1023) abolished collagen binding, whereas mutation of Arg(963) and Arg(1016) reduced collagen binding by 25-35%. These residues are part of loops alpha3beta4 and alpha1beta2 and alpha-helix 3, respectively, and lie near the bottom face of the domain. His(1023) and flanking residues display multiple conformations in available A3-crystal structures, suggesting that binding of A3 to collagen involves an induced-fit mechanism. The collagen-binding site of A3 is located distant from the top face of the domain where collagen-binding sites are found in homologous integrin I domains.

Sequence alignment between vWF and peptides inhibiting the vWF-collagen interaction does not result in the identification of a collagen-binding site in vWF.

We previously found that two peptides (N- and Q-peptide) selected by phage display for binding to an anti-vWF antibody, were able to inhibit vWF-binding to collagen (1). The sequence of those peptides could be aligned with the sequence in vWF at position 1129-1136 just outside the A3-domain. As the peptides represent an epitope or mimotope of vWF for binding to collagen we next wanted to study whether the alignment resulted in the identification of a new collagen binding site in vWF. We mutated the 1129-1136 VWTLPDQC sequence in vWF to VATAPAAC. Expressing this mutant vWF (7.8-vWF) in a fur-BHK cell line resulted in well processed 7.8-vWF containing a normal distribution of molecular weight multimers. However, binding studies of this mutant vWF to rat tail, human and calf skin collagens type 1, to human collagen types III and VI, revealed no decrease in vWF-binding to any of these collagens. Thus, although the N- and Q-peptides did inhibit the vWF-collagen interaction, the resulting alignment with the vWF sequence did not identify a collagen binding site, pointing out that alignments (although with a high percentage of identity) do not always result in identification of binding epitopes. However, suprisingly removal of the A3-domain or changing the vWF sequence at position 1129-1136 resulted in an increase of vWF-binding to human collagen type V1 and to rat tail collagen type 1, implying that these changes result in a different conformation of vWF with an increased binding to these collagens as a consequence.

Binding of von Willebrand factor to collagen type III: role of specific amino acids in the collagen binding domain of vWF and effects of neighboring domains.

Binding of von Willebrand Factor (vWF) to sites of vascular injury is the first step of hemostasis. Collagen types I and III are important binding sites for vWF. We have previously determined the three-dimensional structure of the collagen binding A3 domain of vWF (Huizinga et al., Structure 1997; 5: 1147). We hypothesized that the top face of this domain might be the collagen-binding site. Based on this hypothesis, we made seven vWF mutants (D934A/S936A, V1040A/ V1042A, D1046A, D1066A, D1069A, D1069R, and R1074A). Collagen binding of these mutants was investigated in ELISA and with Surface Plasmon Resonance (BIAcore). In addition, we studied collagen binding of mutants lacking the A2 or D4 domains, which flank the A3 domain. In ELISA, all point mutants and deletion mutants bound to collagen in amounts similar to wild type (WT)-vWF. In the BIAcore we found that WT-vWF has an apparent KD for collagen of 1-7 nM on a subunit base. The apparent kinetic parameters of the point mutants and deletion mutants were not significantly different from WT-vWF, except for DA2-vWF, which had a lower KD. indicating that the A2 domain somehow modulates binding of vWF to collagen type III. Based on our results, we conclude that the amino acid residues mutated by us are not critically involved in the interaction between vWF and collagen type III, which suggests that the collagen binding site is not located on the top face of the A3 domain.

Adhesive domains in the collagen III fragment alpha1(III)CB4 that support alpha2beta1- and von Willebrand factor-mediated platelet adhesion under flow conditions.

Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

von Willebrand factor proteolysis is deficient in classic, but not in bone marrow transplantation-associated, thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) after bone marrow transplantation (BMT) differs from classic TTP in its clinical course and therapy. A characteristic of classic TTP is the inhibition of a plasma protease that specifically cleaves von Willebrand factor (vWF), thus reducing its multimeric size. We investigated whether this protease was also inhibited in BMT-associated TTP. Plasma from patients with classic or BMT-associated TTP was incubated with recombinant vWF R834Q, a vWF mutant with enhanced sensitivity to the protease. The proteolysis of vWF multimers was analyzed and quantified on Western blot. Metalloprotease activity was strongly inhibited in the classic TTP patient group. However, metalloprotease activity was normal in the BMT-associated TTP patient group. The difference in activity between the two patient groups was highly significant (P =.0016). The results indicate that the etiologies of classic and BMT-associated TTP are indeed different and provide an explanation for the lack of success of plasma exchange in BMT-associated TTP.

Crystal structure of the A3 domain of human von Willebrand factor: implications for collagen binding.

BACKGROUND: Bleeding from a damaged blood vessel is stopped by the formation of a platelet plug. The multimeric plasma glycoprotein, von Willebrand factor (vWF), plays an essential role in this process by anchoring blood platelets to the damaged vessel wall under conditions of high shear stress. This factor mediates platelet adhesion by binding both to collagen of the damaged blood vessel and to glycoprotein Ib on the platelet membrane. The A3 domain of vWF allows it to bind to collagen types I and III present in the perivascular connective tissue of the damaged vessel wall. To gain insight into the mechanism of collagen binding by vWF, we have determined the crystal structure of the human vWF A3 domain. RESULTS: The crystal structure of the 20 kDa A3 domain of human vWF (residues 920-1111), determined by the method of multiwavelength anomalous dispersion at 1.8 A resolution, exhibits a common dinucleotide-binding fold. The putative collagen-binding site of the A3 domain is rather smooth and shows a markedly high concentration of negatively charged residues. This region encompasses a potential metal-binding site containing the motif DXSXS, which is required for ligand interaction in the homologous I-type domains of integrins CR3 and LFA-1. Although vWF A3 has considerable sequence and structural similarity with CR3 and LFA-1 in this region, one loop of A3 adopts a conformation which is incompatible with ion binding. CONCLUSIONS: The structure of the A3 domain suggests that adhesion to collagen is primarily achieved through interactions between negatively charged residues on A3 and positively charged residues on collagen. The absence of a pronounced binding groove precludes a large van der Waals surface interaction between A3 and collagen and is consistent with the low affinity for collagen of a single A3 domain and the requirement for multimeric vWF for tight association with collagen. The absence of bound metal ions upon soaking the crystal in MgCl2 and vWF A3's conformational incompatibility for metal binding is consistent with the absence of a functional role for metal ion binding in A3, which contrasts the metal ion activation required for ligand binding by the homologous integrin I type domains.

Active site structure of methylamine dehydrogenase: hydrazines identify C6 as the reactive site of the tryptophan-derived quinone cofactor.

To identify the reactive part of the orthoquinone function of the tryptophan-derived cofactor found in methylamine dehydrogenase (MADH), we have determined the crystal structures of MADH from Thiobacillus versutus inhibited by methylhydrazine and (2,2,2-trifluoroethyl)hydrazine. Extra electron density attached to C6 of the tryptophyl tryptophanquinone cofactor shows that this atom and not C7 is the reactive part of the ortho-quinone moiety. The density retained after hydrazine inhibition is much less extensive than expected, however, suggesting that partial breakdown of the inhibitors after reaction with the cofactor may take place. A detailed description is presented of the cofactor environment in an improved model of MADH which now includes information from the recently determined gene sequence of the cofactor-containing subunit [Ubbink, M., van Kleef, M.A.G., Kleinjan, D., Hoitink, C.W.G., Huitema, F., Beintema, J.J., Duine, J.A., & Canters, G.W. (1991) Eur. J. Biochem. 202, 1003-1012]. We hypothesize that Asp76 is responsible for proton abstraction from the alpha-carbon of the substrate during catalysis.

Three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans determined by molecular replacement at 2.8 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus dentrificans (PD-MADH) has been determined at 2.8 A resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an "X-ray" sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded beta segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 beta-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two covalently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone.

Crystallographic investigations of the tryptophan-derived cofactor in the quinoprotein methylamine dehydrogenase.

A model of tryptophan tryptophylquinone (TTQ), recently proposed by McIntire et al. (Science (1991) 252, 817-824) to be the prosthetic group of the quinoprotein methylamine dehydrogenase, has been compared with electron density maps of this dehydrogenase from Thiobacillus versutus and Paracoccus denitrificans. The comparison shows that the TTQ model can be neatly accommodated, providing strong supportive evidence that TTQ is indeed the cofactor for this group of quinoproteins.