Katsunori Horii

A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms

Hospital-acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains; however, the precise role of these proteins in biofilm formation is unclear. We show here, using analytical ultracentrifugation (AUC) and circular dichroism (CD), that G5 domains from Aap are zinc (Zn2+)-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn2+, incorporating 2–3 Zn2+ ions in the dimer interface. Tandem G5 domains associate in a modular fashion, suggesting a “zinc zipper” mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. We demonstrate, using a biofilm plate assay, that Zn2+ chelation specifically prevents biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibit biofilm formation in a dose-dependent manner. Thus, the complex three-dimensional architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. Zn2+ chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.

Crystal structure of trimestatin, a disintegrin containing a cell adhesion recognition motif RGD.

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet-platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7A resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin alphaVbeta3 suggests that the Arg binds to the propeller domain, and Asp to the betaA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer.

Convulxin Forms a Dimer in Solution and Can Bind Eight Copies of Glycoprotein VI: Implications for Platelet Activation†

Convulxin (CVX) is a C-type lectin-like protein from the venom of the South American rattlesnake that functions as a potent agonist of the platelet collagen receptor glycoprotein VI (GPVI). Although CVX is widely used as a platelet agonist, the molecular basis for its extremely high potency is not clear. In order to delineate possible mechanisms for CVX-induced GPVI activation, we used analytical ultracentrifugation to determine the assembly state of CVX in solution and surface plasmon resonance in order to understand the affinity, kinetics, and stoichiometry of GPVI binding to CVX. We show here that CVX exists in solution as a dimer of alpha4beta4 rings, yielding eight potential binding sites for GPVI. Binding studies confirm that all eight sites are able to bind GPVI tightly, each with high picomolar or low nanomolar affinity. Reanalysis of previously determined crystal structures of CVX revealed the dimer in both structures. The dimeric nature of CVX and its ability to bind eight GPVI molecules suggest that it might be capable of binding to GPVI expressed on two opposing surfaces. Agglutination assays using GPVI-coated beads confirm that CVX is able to bridge distinct GPVI-coated surfaces and indicate that CVX agglutination of platelets is dependent on GPVI binding. Thus, in addition to clustering up to eight GPVI receptors, CVX may facilitate platelet activation by bridging platelets directly.

Folding-unfolding of goat ?-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations

Folding reaction of goat α‐lactalbumin has been studied by stopped‐flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the α‐domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native‐state stability of goat α‐lactalbumin against the guanidine hydrochloride‐induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild‐type and mutant goat α‐lactalbumin measured by stopped‐flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high‐temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter‐residue contacts and with the water molecule penetration in the 400‐K simulation as well as in four of the six 600‐K simulations. Disruption of the N‐terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report. Proteins 2001;42:49–65.

Calcium-dependent Dimerization of Human Soluble Calcium Activated Nucleotidase CHARACTERIZATION OF THE DIMER INTERFACE

Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile170, Ser172, and Ser226 with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys30, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.

Contribution of Thr29 to the thermodynamic stability of goat alpha-lactalbumin as determined by experimental and theoretical approaches.

The Thr29 residue in the hydrophobic core of goat α‐lactalbumin (α‐LA) was substituted with Val (Thr29Val) and Ile (Thr29Ile) to investigate the contribution of Thr29 to the thermodynamic stability of the protein. We carried out protein stability measurements, X‐ray crystallographic analyses, and free energy calculations based on molecular dynamics simulation. The equilibrium unfolding transitions induced by guanidine hydrochloride demonstrated that the Thr29Val and Thr29Ile mutants were, respectively, 1.9 and 3.2 kcal/mol more stable than the wild‐type protein (WT). The overall structures of the mutants were almost identical to that of WT, in spite of the disruption of the hydrogen bonding between the side‐chain OH group of Thr29 and the main‐chain CO group of Glu25. To analyze the stabilization mechanism of the mutants, we performed free energy calculations. The calculated free energy differences were in good agreement with the experimental values. The stabilization of the mutants was mainly caused by solvation loss in the denatured state. Furthermore, the OH group of Thr29 favorably interacts with the CO group of Glu25 to form hydrogen bonds and, simultaneously, unfavorably interacts electrostatically with the main‐chain CO group of Thr29. The difference in the free energy profile of the unfolding path between WT and the Thr29Ile mutant is discussed in light of our experimental and theoretical results. Proteins 2001;45:16–29.