Search

Erythropoietin receptor (EPOR) is thought to be activated by ligand-induced homodimerization. However, structures of agonist and antagonist peptide complexes of EPOR, as well as an EPO-EPOR complex, have shown that the actual dimer configuration is critical for the biological response and signal efficiency. The crystal structure of the extracellular domain of EPOR in its unliganded form at 2.4 angstrom resolution has revealed a dimer in which the individual membrane-spanning and intracellular domains would be too far apart to permit phosphorylation by JAK2. This unliganded EPOR dimer is formed from self-association of the same key binding site residues that interact with EPO-mimetic peptide and EPO ligands. This model for a preformed dimer on the cell surface provides insights into the organization, activation, and plasticity of recognition of hematopoietic cell surface receptors.

The crystal structure of a complex between a 24-amino acid peptide from the third variable (V3) loop of human immunodeficiency virus-type 1 (HIV-1) gp 120 and the Fab fragment of a broadly neutralizing antibody (59.1) was determined to 3 angstrom resolution. The tip of the V3 loop containing the Gly-Pro-Gly-Arg-Ala-Phe sequence adopts a double-turn conformation, which may be the basis of its conservation in many HIV-1 isolates. A complete map of the HIV-1 principal neutralizing determinant was constructed by stitching together structures of V3 loop peptides bound to 59.1 and to an isolate-specific (MN) neutralizing antibody (50.1). Structural conservation of the overlapping epitopes suggests that this biologically relevant conformation could be of use in the design of synthetic vaccines and drugs to inhibit HIV-1 entry and virus-related cellular fusion.

The crystal structure of the Fab fragment of a human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody Fab has been determined at 2.8 A resolution in complex with a linear 16-residue peptide from the third hypervariable region (V3) of gp120. The first 9 residues of the peptide are ordered in the electron density maps, and their conformation is in partial agreement with the beta-strand-type II beta-turn structure predicted for this portion of the V3 loop. Notably, several of the peptide residues that are well conserved among different HIV-1 isolates contact a nonpolar 25-A-long groove in the antibody-combining site. The largely extended structure of the peptide differs from the beta-turns seen as the primary determinants in other published anti-peptide Fab structures. Analysis of the specific Fab-peptide interactions only partially explains the MN isolate specificity shown by this antibody.

X-ray quality crystals of an Fab fragment from an antipeptide monoclonal antibody (R/V3-50.1) that recognizes the principal neutralizing determinant (PND) of the gp120 glycoprotein of human immunodeficiency virus type 1 (HIV-1) (MN isolate) were grown as uncomplexed and peptide complexed forms. Crystals of the free Fab grew from high salt in orthorhombic space groups P2(1)2(1)2(1) and I222 and from polyethylene glycol in space groups P1 and P2(1). Seeds from either the P1 and P2(1) native (uncomplexed) Fab crystals induced nucleation of crystals of the Fab complexed to a 16-residue synthetic peptide corresponding to the PND when streak seeded into preequilibrated solutions of this complex. Data were collected from these complex crystals and from each of the four native Fab forms to at least 2.8 A resolution. The genes for the variable domain of the Fab were cloned and sequenced and the primary amino acid sequence was deduced from this information. Knowledge of the three-dimensional structure of this Fab-peptide complex will be important in the understanding of the PND of HIV-1 and its recognition by neutralizing monoclonal antibodies.

Crystals of the Fab' fragment from the monoclonal anti-peptide antibody B1312 and of the Fab'-peptide antigen complex have been characterized. The monoclonal antibodies were raised against a synthetic homologue of the C-helix of myohemerythrin (residues 69-87 in myohemerythrin). The Fab'-peptide complex crystallizes in space group P6322 with unit cell dimensions a = b = 142.5 A, c = 101.5 A, alpha = beta = 90 degrees, gamma = 120 degrees, and Z = 1. The native Fab' crystallizes in space group P212121 with unit cell dimensions a = 98.0 A, b = 151.7 A, c = 80.8 A, alpha = beta = gamma = 90 degrees, and Z = 2. Both crystal forms diffract to beyond 2.6 A resolution. We also report the cDNA and predicted amino acid sequences for the variable regions of both the light and heavy chains of this anti-peptide antibody.