X-RAY STRUCTURES OF A DESIGNED BINDING SITE IN TRYPSIN-ECOTIN COMPLEX SHOW METAL-DEPENDENT GEOMETRY Linda S. Brinen^{[[daggerdbl]]}, W. Scott Willett^[[section]], Charles S. Craik^[[section]] and Robert J. Fletterick^{[[daggerdbl]]}. ^{[[daggerdbl]]}Department of Biochemistry and Biophysics, ^[[section]]Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94143

The three-dimensional structures of complexes of trypsin N143H, E151H bound to ecotin A86H are determined at 2.0 Å resolution via X-ray crystallography in the absence and presence of the transition metals Zn²⁺, Ni²⁺, and Cu²⁺. The binding site for these transition metals was constructed by substitution of key amino acids with histidine at the trypsin-ecotin interface in the S2'/P2' pocket. Three histidine side chains, two on trypsin at positions 143 and 151, and one on ecotin at position 86, anchor the metals and provide extended catalytic recognition for substrates with His in the P2' pocket. Comparisons of the three-dimensional structures show the different geometries that result upon the binding of metal in the engineered tridentate site and suggest a structural basis for the kinetics of the metal-regulated catalysis. The structural results indicate that the geometry of the engineered metal binding site is dictated by preferred geometry of the metal ion and not the structural constraints of the surrounding protein. This finding, which could not be adequately predicted by modeling studies done on this system, is key to the design of a metal binding site. Of the three metals, the binding of zinc results in the most favorable binding geometry, not dissimilar to those observed in naturally occurring zinc-binding proteins. This work represents the first successful X-ray crystallographic investigation of a de novo engineered metal binding site in the absence and presence of metal ions.