Receptor rigidity and ligand mobility in trypsin–ligand complexes

The trypsin-like serine proteases comprise a structurally similar family of proteins with a wide diversity of biological functions. Mem- bers of this family play roles in digestion, hemosta- sis, immune responses, and cancer metastasis. Bo- vine trypsin is an archetypical member of this family that has been extensively characterized both func- tionally and structurally, and that preferentially hydrolyzes Arg/Lys-Xaa peptide bonds. We have used molecular dynamics (MD) simulations to study bovine trypsin complexed with the two noncovalent small-molecule ligands, benzamidine and tranyl- cypromine, that have the same hydrogen-bond do- nating moieties as Arg and Lys side-chains, respec- tively. Multiple (10) simulations ranging from 1 ns to 2.2 ns, with explicit water molecules and periodic boundary conditions, were performed. The simula- tions reveal that the trypsin binding pocket resi- dues are relatively rigid regardless of whether there is no ligand, a high-affinity ligand (benzamidine), or a low-affinity ligand (tranylcypromine). The ther- mal average of the conformations sampled by benza- midine bound to trypsin is planar and consistent with the planar internal geometry of the benzami- dine crystallographic model coordinates. However, the most probable bound benzamidine conforma- tions are 25° out of plane, implying that the ob- served X-ray electron density represents an average of densities from two mirror symmetric, nonplanar conformations. Solvated benzamidine has free en- ergy minima at 45°, and the induction of a more planar geometry upon binding is associated with 1 kcal/mol of intramolecular strain. Tranylcypro- mine's hydrogen-bonding pattern in the MD differs substantially from that inferred from the X-ray elec- tron density. Early in simulations of this system, tranylcypromine adopts an alternative binding con- formation, changing from the crystallographic con- formation, with a direct hydrogen bond between its amino moiety and the backbone oxygen of Gly219, to one having a bridging water molecule. This result is consistently seen with the CHARMM22, Amber, or OPLS-AA force fields. The trypsin-tranylcypromine hydrogen-bonding pattern observed in the simula- tions also occurs as the crystallographic binding mode of the Lys15 side-chain of bovine pancreatic trypsin inhibitor bound to trypsin. In this latter cocrystal, a bridging crystallographic water does reside between the side-chain's amino group and the trypsin Gly219 backbone oxygen. Furthermore, the trypsin-tranylcypromine simulations sample two different stable noncrystallographic binding poses. These data suggest that some of the electron density ascribed to tranylcypromine in the X-ray model is rather due to a bound water molecule, and that multiple tranylcypromine binding conformations (crystallographic disorder) may be the cause of ambiguous electron density. The combined trypsin- benzamidine and trypsin- tranylcypromine results highlight the ability of simulations to augment pro- tein-ligand complex structural data by deconvolut- ing the effects of thermal and structural averaging, and by finding energetically optimal ligand and bound water positions for weakly bound ligands. Proteins 2005;58:407- 417. © 2004 Wiley-Liss, Inc.