Search

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69–74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.

Based on experimental NMR data, a model was generated for the conformation of the disulfide-bond-closed cyclic peptide corresponding to the whole V3 loop of the consensus HIV-1 strain in a 20% trifluoroethanol/water solution. The obtained family of structures shows a prominent and well-defined amphipathic alpha helix at the C-terminal end of the peptide from Thr23 to Gln32. A series of turns characterizes the central Gly15-Tyr21 region, while the N-terminal region is poorly defined. Independent experimental data confirms the features of this model, and suggests that this type of conformation can be readily adopted when the V3 loop is in contact with a membrane. The examined V3 loop belongs to a macrophage tropic strain, and using the model, a structural explanation is proposed for the different requirements of V3 loops belonging to macrophage and T-cell line tropic HIV-1 strains.

Rs-AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs-AFPs have been isolated (Rs-AFP1-4). The structure of Rs-AFP1 consists of three beta-strands and an alpha-helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure-function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs-AFP2 is concentrated mainly in the beta2-beta3 loop. In this study, we synthesized linear 19-mer peptides, spanning the entire beta2-beta3 loop, that were found to be almost as potent as Rs-AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19-mer loop peptides, cysteines can be replaced by alpha-aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19-mer peptides, forced to adopt a hairpin structure by the introduction of one or two non-native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19-mer peptides, like Rs-AFP2 itself, cause increased Ca2+ influx in pregerminated fungal hyphae.

The disulfide-bridge-closed cyclic peptide corresponding to the whole V3 loop of the RF HIV-1 strain was examined by proton two-dimensional NMR spectroscopy in water and water/trifluoroethanol solutions. Although most of the peptide is conformationally averaged in water, the NOE data support a beta-turn conformation for the central conservative GPGR region and the presence of nascent helix. Upon addition of trifluoroethanol, helix formation in the C-terminal part becomes apparent. This is confirmed by CD data. NOEs indicative of multiple and transient beta-turns around the Asn6 glycosylation site and NOEs fitting X-ray data on a linear V3 peptide-Fab complex also emerge. The C-terminal helix is shown to have amphipathic character and might thus assist in the infection process.

The disulfide bridge closed cyclic peptide corresponding to the whole Consensus V3 loop of the envelope protein gp120 of HIV-1 was examined by proton 2D-NMR spectroscopy in water and in a 20% trifluoroethanol/water solution. In water, NOE data support a β-turn conformation for the central conservative GPGR region and point towards partial formation of a helix in the C-terminal part. Upon addition of trifluoroethanol, a C-terminal helix is formed. This is evidenced by NOE data, α-proton chemical shift changes and changes in the J N α vicinal coupling constants. The C-terminal helix is amphipathic and also occurs in other examined strains. It could therefore be an important feature for the functioning of the V3 loop.