Background: Nicotinic acetylcholine receptors are activated by agonists at an orthosteric site and modulated by ligands at allosteric sites. Results: We identify amino acids required for the coupling between orthosteric and allosteric sites. Conclusion: Allosteric activation can occur even when the orthosteric binding site is nonfunctional. Significance: Insights are provided into the cooperative functions of orthosteric and allosteric activators of the ␣7 nAChR.

The humanized monoclonal antibody HzKR127 recognizes the preS1 domain of the human hepatitis B virus surface proteins with a broadly neutralizing activity in vivo. We present the crystal structures of HzKR127 Fab and its complex with a major epitope peptide. In the complex structure, the bound peptide forms a type IV ␤-turn followed by 310 helical turn, the looped-out conformation of which provides a structural basis for broad neutralization. Upon peptide binding, the antibody undergoes a dramatic complementarity determining region H3 lid opening. To understand the structural implication of the virus neutralization, we carried out comprehensive alanine-scanning mutagenesis of all complementarity determining region residues in HzKR127 Fab. The functional mapping of the antigen-combining site demonstrates the specific roles of major binding determinants in antigen binding, contributing to the rational design for maximal humanization and affinity maturation of the antibody.alanine-scanning mutagenesis ͉ crystal structure ͉ humanized antibody ͉ virus neutralization H uman hepatitis B virus (HBV) is a small enveloped DNA virus that causes acute and chronic hepatitis in humans (1-3). There are approximately 400 million carriers of HBV worldwide, posing a serious health threat. The current HBV vaccines have several limitations, including nonresponsiveness, escape mutants, and low immunogenicity (4). Recently, the potential of immunoprophylaxis for virus prevention has been increased by the discovery of monoclonal antibodies that have broad specificity toward various isolates of infectious viruses (5, 6).The HBV envelope consists of three surface glycoproteins called the large (L), middle (M), and small (S) proteins. All of these proteins are translated from a single ORF comprising preS1, preS2, and S domains (7). Translation of S, preS2ϩS, and preS1ϩpreS2ϩS domains results in S, M, and L proteins, respectively (Fig. 1). The L protein, preferentially localized in infectious HBV particles, plays an essential role in viral infection (7,8), and its preS1 domain is responsible for cell attachment in viral infection (9-12). In particular, the residue 21-47 region of preS1 has been suggested to contain a specific binding site for hepatocyte receptors and also has been shown to carry virusneutralizing epitopes (10-13).In the blood of HBV-infected persons, the noninfectious subviral particles with M and S proteins are present in excess amounts over the infectious virus particles containing L protein (7,8). Thus, neutralizing anti-preS1 antibodies are thought to have advantages over the neutralizing antibodies targeting S protein, which are currently undergoing clinical trials (4). Previously, we generated an anti-preS1 murine antibody KR127 that recognizes adr subtype preS1 (residues 37-45) (15). KR127 and the humanized version of KR127 antibody (HzKR127) bind to the preS1 domain of various clinical HBV isolates, including both adr and ayw subtypes, suggesting their broad neutralizing activity (15, 16). HzKR127 also has been sh...

Dihydrohpoamide acetyl transferase (E2), a catalytic and structural component of a multienzyme complex that catalyzes the oxidative decarboxylation of pyruvate, forms the central core to which the other components are bound. We have utilized protein engineering and 3-D electron microscopy to study the structural organization of the largest multienzyme complex known (Mr ∼ 107). The structures of the truncated 60-mer core (tE2) and complexes of the tE2 associated with a binding protein (BP), and the BP associated with its dihydrohpoamide dehydrogenase (BP'E3) and the intact E2 associated with BP and the pyruvate dehydrogenase (E1) were determined (Figs. 1 and 2). The tE2 core is a pentagonal dodecahedron consisting of 20 cone-shaped trimers interconnected by 30 bridges.Previous studies have given rise to the generally accepted belief that BP and BP'E3 components are bound on the outside of the E2 scaffold and that E1 is similarly bound to the core in variable positions by flexible tethers.

The dihydrolipoamide dehydrogenase-binding protein (E3BP), a component of the Saccharomyces cerevisiae and mammalian pyruvate dehydrogenase (PDH) complexes, anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2). To gain further insight into the number and localization of binding sites for E3BP on the 60-mer E2, truncated forms of the E3BP lacking the lipoyl and E3-binding domains were engineered by deletion mutagenesis. The recombinant proteins contained a polyhistidine extension on the amino terminus to facilitate purification to near-homogeneity. The stoichiometry of binding of the truncation mutants to a truncated form (inner core) of E2 (tE2, residues 181-454), lacking the lipoyl domain and the E1-binding domain, was determined. Mixtures containing tE2 and excess intact or truncated forms of E3BP were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or tE3BP, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that tE2 binds about 20 copies of intact E3BP-H, about 24 copies of tE3BP-H144 (residues 144-380), lacking the lipoyl domain, and about 31 copies of tE3BP-H218 (residues 218-380), lacking both the lipoyl and E3-binding domains. The results indicate that there apparently is a binding site for E3BP on each E2 subunit and that steric hindrance by segments of E3BP prevents full stoichiometric binding of E3BP to the pentagonal dodecahedron-like E2.

No abstract