NMR three-dimensional solution structure of the serine protease inhibitor cyclotheonamide A

The nmr solution conformation of cyclotheonamide A (CtA) was determined in aqueous media. The data produced 15 distance and 10 torsional constraints which were used to generate conformations using restrained simulated annealing (SA) and distance geometry/simulated annealing (DG/SA) calculations. Two different calculation protocols were performed to ensure proper sampling of conformational space and even though the torsional restraints were input differently, both calculation methods yielded the same conformation of CtA. In the structure calculations, all solutions of the Karplus equation were sampled simultaneously using the restrained SA protocol and large ranges were used for the dihedral restraints in the DG/SA protocol because all solutions to the Karplus equation could not be sampled simultaneously. The solution conformation was also compared to the solid state x‐ray conformations of CtA bound to thrombin and trypsin. The conformation of the residues important for active site binding (d‐Phe, h‐Arg, and Pro) are nearly identical in aqueous solution and solid state with largest differences at the a‐Ala and v‐Tyr residues. CtA appears to be preordered in structure and does not undergo a significant conformational change upon binding to the enzyme active site. © 1997 John Wiley & Sons, Inc.