Oligomer-specific conformations of the human immunodeficiency virus (HIV-1) gp41 envelope glycoprotein ectodomain recognized by human monoclonal antibodies.

Trimerization of the human immunodeficiency virus (HIV-1) envelope glycoproteins is mediated by the ectodomain of the gp41 transmembrane glycoprotein. Here we investigate oligomer-specific conformations of gp41 by using monoclonal antibodies (MAbs) from HIV-1-infected humans. Human MAbs directed against the cluster I region of gp41 recognized trimeric, dimeric, and monomeric forms of soluble envelope glycoproteins; thus, the integrity of the cluster I epitopes is minimally affected by the oligomeric state. In contrast, human MAbs to the cluster II region were all oligomers specific. One cluster II MAb, 126-6, recognized exclusively the trimeric form of envelope glycoproteins, whereas the others recognized both trimeric and dimeric forms. Thus, a distinct trimer-specific conformation exists in the cluster II region of gp41. Analysis of soluble envelope glycoprotein mutants revealed that gp41 sequences immediately N-terminal to isoleucine 646 contribute to the formation of both the trimer and the trimer-specific conformational epitope.

The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA.

The DH-PH domain tandems of Dbl-homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho-family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH-PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH-PH tandem of RhoA-specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH-PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide-free RhoA exhibits elevated dynamics when in complex with DH-PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.