Wim Vranken

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.

An NMR-based identification of peptide fragments mimicking the interactions of the cathepsin B propeptide

Selected fragments of the 62‐residue proregion (or residues 1p–62p) of the cysteine protease cathepsin B were synthesized and their interactions with cathepsin B studied by use of proton NMR spectroscopy. Peptide fragments 16p–51p and 26p–51p exhibited differential perturbations of their proton resonances in the presence of cathepsin B. These resonance perturbations were lost for the further truncated 36p–51p fragment, but remained in the 26p–43p and 28p–43p peptide fragments. Residues 23p–26p or TWQ25A in the N‐terminal 1p–29p fragment did not show cathepsin B‐induced resonance perturbations although the same residues had strongly perturbed proton resonances within the 16p–51p peptide. Both the 1p–29p and 36p–51p fragments lack a common set of hydrophobic residues 30p–35p or F30YNVDI35 from the proregion. The presence of residues F30YNVDI35 appears to confer a conformational preference in peptide fragments 16p–51p, 26p–51p, 28p–43p and 26p–43p, but the same residues induce the aggregation of peptides 16p–36p and 1p–36p. The peptide fragment 26p–43p binds to the active site, as indicated by its inhibition of the catalytic activity of cathepsin B. The cathepsin B prosegment can therefore be reduced into smaller, but functional subunits 28p–43p or 26p–43p that retain specific binding interactions with cathepsin B. These results also suggest that residues F30YNVDI35 may constitute an essential element for the selective inhibition of cathepsin B by the full‐length cathepsin B proregion.