Eric G. Huizinga

Structures of complement component C3 provide insights into the function and evolution of immunity

The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 (1,641 residues) is central to the three complement pathways and results in inflammation and elimination of self and non-self targets. Here we present crystal structures of native C3 and its final major proteolytic fragment C3c. The structures reveal thirteen domains, nine of which were unpredicted, and suggest that the proteins of the α2-macroglobulin family evolved from a core of eight homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the α-chain, including movement of a critical interaction site through a ring formed by the domains of the β-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3.

An experimental model to study the in vivo survival of von Willebrand factor. Basic aspects and application to the R1205H mutation.

To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t½α = 13 min) and a slow secondary phase (t½β = 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of full-length VWF to truncated variants. Deletion of both the amino-terminal D′-D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D′-D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT = 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT = 4.5 h, p < 0.02). These results point to a role of the D′-D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT = 0.3 h; p = 0.004). Therefore, this mutation seems to abrogate the protective effect of the D′-D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.

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 A3s 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.

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombin-cleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors.

Factor B structure provides insights into activation of the central protease of the complement system

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-Å resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion–dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices. Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complement-amplification step in the immune response.

Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution

The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer–monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.

Calcium modulates force sensing by the von Willebrand factor A2 domain.

von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing.

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.

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

A model of tryptophan tryptophylquinone (TTQ), recently proposed by Mclntire 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.

Implications for collagen I chain registry from the structure of the collagen von Willebrand factor A3 domain complex.

Fibrillar collagens, the most abundant proteins in the vertebrate body, are involved in a plethora of biological interactions. Plasma protein von Willebrand factor (VWF) mediates adhesion of blood platelets to fibrillar collagen types I, II, and III, which is essential for normal haemostasis. High affinity VWF-binding sequences have been identified in the homotrimeric collagen types II and III, however, it is unclear how VWF recognizes the heterotrimeric collagen type I, the superstructure of which is unknown. Here we present the crystal structure of VWF domain A3 bound to a collagen type III-derived homotrimeric peptide. Our structure reveals that VWF-A3 interacts with all three collagen chains and binds through conformational selection to a sequence that is one triplet longer than was previously appreciated from platelet and VWF binding studies. The VWF-binding site overlaps those of SPARC (also known as osteonectin) and discodin domain receptor 2, but is more extended and shifted toward the collagen amino terminus. The observed collagen-binding mode of VWF-A3 provides direct structural constraints on collagen I chain registry. A VWF-binding site can be generated from the sequences RGQAGVMF, present in the two α1(I) chains, and RGEOGNIGF, in the unique α2(I) chain, provided that α2(I) is in the middle or trailing position. Combining these data with previous structural data on integrin binding to collagen yields strong support for the trailing position of the α2(I) chain, shedding light on the fundamental and long-standing question of the collagen I chain registry.