Franky Fant

Mutational Analysis of a Plant Defensin from Radish (Raphanus sativus L.) Reveals Two Adjacent Sites Important for Antifungal Activity

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI β-turn connecting β-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting β-strand 1 and the α-helix and contiguous residues on the α-helix and β-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.

The three-dimensional solution structure of Aesculus hippocastanum antimicrobial protein 1 determined by 1H nuclear magnetic resonance.

Aesculus hippocastanum antimicrobial protein 1 (Ah‐AMP1) is a plant defensin isolated from horse chestnuts. The plant defensins have been divided in several subfamilies according to their amino acid sequence homology. Ah‐AMP1, belonging to subfamily A2, inhibits growth of a broad range of fungi. So far, a three‐dimensional structure has been determined only for members of subfamilies A3 and B2. In order to understand activity and specificity of these plant defensins, the structure of a protein belonging to subfamily A2 is needed. We report the three‐dimensional solution structure of Ah‐AMP1 as determined from two‐dimensional 1H nuclear magnetic resonance data. The structure features all the characteristics of the “cysteine‐stabilized αβ‐motif.”A comparison of the structure, the electrostatic potential surface and regions important for interaction with the fungal receptor, is made with Rs‐AFP1 (plant defensin of subfamily A3). Thus, residues important for activity and specificity have been assigned. Proteins 1999;37:388–403. ©1999 Wiley‐Liss, Inc.

The complete Consensus V3 loop peptide of the envelope protein gp120 of HIV-1 shows pronounced helical character in solution

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 Nα 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.

Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements.

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.

Structural characterization by NMR of rifabutinol, a derivative of rifabutin.

Rifabutin, a naphthalenic ansamycin, is a specific inhibitor of bacterial DNA‐directed RNA polymerase and inhibits in vitro the replication of HIV‐1. The synthesis of rifabutinol by PtO2–NaBH4 reduction of rifabutin in ethanol is described. Full assignments in CDCl3 are presented of the 1H and 13C NMR resonance signals using two‐dimensional homonuclear and heteronuclear correlation techniques and selective proton decoupling methods. The three‐dimensional structure of the ansa‐chain of rifabutinol was calculated from NOE contacts and vicinal coupling constants, using a simulated annealing protocol starting from random coordinates. Measurements of the 13C T1 relaxation times of rifabutin in solution are consistent with a rigid ansa‐chain. Copyright