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The solution structure of the hepatitis C virus (BK strain) NS3 protein Nterminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zincbinding site exposed on the surface, subject to a slow conformational exchange process.

A tricyclic ketone with seven stereogenic centers produced in >95:5 diastereomeric excess by an asymmetric Diels-Alder reaction has been subjected to a careful 2-D NMR(CDCl 3 )/MD analysis by Reggelin and co-workers and interpreted in terms of a single conformation in CDCl 3 solution. The present work examines the validity of this interpretation. Specifically, the conformational profile of the endo isomer of the tricyclic ketone was examined by Monte Carlo searches with both the MM2* and MMFF force fields in MacroModel using the GB/SA CHCl 3 solvent continuum model. The two sets of conformations were then combined and optimized with the MM3(96) force field. The structures of the resulting conformations and the NMR-derived geometric variables were together subjected to a NAMFIS analysis (NMR analysis of molecular flexibility in solution). Ideally, the procedure deconvolutes the thermally averaged NMR data into a small family of conformations that optimally represents the J-derived torsions and the NOE-derived distances. It is concluded that the tricyclic ketone under study is not well-characterized by a single conformation in CDCl 3 solution, but by a set of rapidly equilibrating conformers that produces a deceptively averaged NMR spectrum. It would appear that the previously suggested structure is a virtual conformation obligated to compress multiconformer features into a single 3-D construct. In additon, we find that current force fields do not uniformly represent the conformational profile of polar molecules such as 1. The cause is traced primarily to the variable treatment of electrostatic effects.

Cerato-platanin (CP) is a secretion protein produced by the fungal pathogen Ceratocystis platani, the causal agent of the plane canker disease and the first member of the CP family. CP is considered a pathogen-associated molecular pattern because it induces various defense responses in the host, including production of phytoalexins and cell death. Although much is known about the properties of CP and related proteins as elicitors of plant defense mechanisms, its biochemical activity and host target(s) remain elusive. Here, we present the three-dimensional structure of CP. The protein, which exhibits a remarkable pH and thermal stability, has a double ␤-barrel fold quite similar to those found in expansins, endoglucanases, and the plant defense protein barwin. Interestingly, although CP lacks lytic activity against a variety of carbohydrates, it binds oligosaccharides. We identified the CP region responsible for binding as a shallow surface located at one side of the ␤-barrel. Chemical shift perturbation of the protein amide protons, induced by oligo-N-acetylglucosamines of various size, showed that all the residues involved in oligosaccharide binding are conserved among the members of the CP family. Overall, the results suggest that CP might be involved in polysaccharide recognition and that the double ␤-barrel fold is widespread in distantly related organisms, where it is often involved in host-microbe interactions.