Solution structure of a calmodulin-binding domain in the carboxy-terminal region of HIV type 1 gp160.

The cytosolic domain of human immunodeficiency virus gp160 glycoprotein contains two calmodulin-binding regions. The role of these domains in modulating intracellular calmodulin signaling is of considerable interest in unraveling the mechanism whereby calmodulin regulates Fas-mediated apoptosis in HIV-infected cells. In this investigation we have employed 2D-NMR spectroscopy to determine the solution structure of the 30-residue calmodulin-binding domain corresponding to residues 826-855 of gp160. In solution, the gp160 (826-855) peptide exhibits a high degree of segmental flexibility. Within its conformational manifold, we have detected two separate flexible amphipathic helices involving residues 826-841 and 846-855 connected by a highly flexible type-II beta-turn at Pro-843 and Arg-844. The observed NOE pattern as well as the observation of long-range NOE contacts between the side chains of His-841 and Ile-846 are compatible with the presence of this turn in the conformational manifold of this peptide. This investigation focusing on the properties of the free peptide in solution paves the way for extending the investigations on the interaction of calmodulin with HIV-1 gp160.

Association of a model class A (apolipoprotein) amphipathic alpha helical peptide with lipid: high resolution NMR studies of peptide.lipid discoidal complexes.

Class A amphipathic helical peptides have been shown to mimic apolipoprotein A-I, the major protein component of high density lipoproteins and have been shown to inhibit atherosclerosis in several dyslipidemic mouse models. Previously we reported the NMR structure of Ac-18A-NH2, the base-line model class A amphipathic helical peptide in a 50% (v/v) trifluoroethanol-d3/water mixture, a membrane-mimic environment (Mishra, V. K., Palgunachari, M. N., Anantharamaiah, G. M., Jones, M. K., Segrest, J. P., and Krishna, N. R. (2001) Peptides 22, 567-573). The peptide Ac-18A-NH2 forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Because subtle structural changes in the peptide.lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of recombinant peptide.1,2-dimyristoyl-sn-glycero-3-phosphocholine complexes. The peptide adopts a well defined amphipathic alpha helical structure in association with the lipid at a 1:1 peptide:lipid weight ratio. Nuclear Overhauser effect spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. The pattern of observed peptide-lipid nuclear Overhauser effects is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). Based on the results of chemical cross-linking and molecular modeling, we propose that peptide helices are arranged in a head to tail fashion to cover the edge of the disc. This arrangement of peptides is also consistent with the pKa values of the Lys residues determined previously. Taken together, these results provide for the first time a high resolution structural view of the peptide.lipid discoidal complexes formed by a class A amphipathic alpha helical peptide.