High-resolution structure of bovine pancreatic trypsin inhibitor with altered binding loop sequence

A mutant of bovine pancreatic trypsin inhibitor (BPTI) has been constructed and expressed in Escherichia coli in order to probe the kinetic and struct ural consequences of truncating the binding loop residues to alanine. in ad dition to two such mutations (Thr11Ala and Pro13Ala), it has a conservative Lys15Arg substitution at position P-1 and an unrelated Met52Leu change. In spite of the binding loop modification, the affinity for trypsin is only 3 0 times lower than that of the wild-type protein. At pH 7.5 the protein can be crystallized on the time-scale of hours, yielding very stable crystals of a new (tetragonal) form of BPTI. Conventional source X-ray data collecte d to 1.4 Angstrom at room temperature allowed anisotropic structure refinem ent characterized by R = 0.1048. The structure reveals all 58 residues, inc luding the complete C terminus, which is in a salt-bridge contact with the N terminus. The Cys14-Cys38 disulfide bridge is observed in two distinct ch iralities. This bridge, together with an internal water molecule, contribut es to the stabilization of the binding loop. The Ala mutations have only an insignificant and localized effect on the binding loop, which retains its wild-type conformation (maximum deviation of loop C-alpha atoms of 0.7 Angs trom at Ala13). Four (instead of the typical three) additional water molecu les are buried in an internal cleft and connected to the surface via a sulf ate anion. Three more SO42- anions are seen in the electron density, one of them located on a 2-fold axis. It participates in the formation of a dimer ic structure between symmetry-related BPTI molecules, in which electrostati c and hydrogen bonding interactions resulting from the mutated Lys15Arg sub stitution are of central importance. This dimeric interaction involves dire ct recognition loop-recognition loop contacts, part of which are hydrophobi c interactions of the patches created by the alanine mutations. Another 2-f old symmetric interaction between the BPTI molecules involves the formation of an antiparallel intermolecular beta-sheet that, together with the adjac ent intramolecular beta-hairpin loops, creates a four-stranded structure. ( C) 2000 Academic Press.