Substitutions at the P 1 0 Position in BPTI Strongly Affect the Association Energy with Serine Proteinases Agnieszka Grzesiak 1 , Ronny Helland 2 , Arne O. Smala Ês 2 Daniel Krowarsch 1 , Michal Dadlez 3 and Jacek Otlewski 1 * 1 Protein Engineering Laboratory, Institute of Biochemistry and Molecular Biology, University of Wroclaw Tamka 2, 50-137, Wroclaw Poland 2 Protein Crystallography Group, Department of Chemistry, University of Tromsù, N-9037, Tromsù Norway 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5A 02-106, Warszawa, Poland The role of the S 1 0 subsite in trypsin, chymotrypsin and plasmin has been examined by measuring the association with seven different mutants of bovine pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, Val, Leu, Arg, and Trp at the P 1 0 position of the reactive site. The effects of substitutions at the P 1 0 position on the association constants are very large, comprising seven orders of magnitude for trypsin and plas- min, and over ®ve orders for chymotrypsin. All mutants showed a decrease of the association constant to the three proteinases in the same order: Ala > Gly > Ser > Arg > Val > Leu > Trp. Calorimetric and circular dichroism methods showed that none of the P1 0 substitutions, except the P1 0 -Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of the complex formed between bovine b-trypsin and P 1 0 -Leu BPTI showed that the P 1 0 -Leu sterically con¯icts with the side- chain of P 3 0 -Ile, which thereby is forced to rotate approximately 90  . Ile18 (P 3 0 ) in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Introduction of a large side-chain at the P1 0 position apparently leads to a cascade of small alterations of the trypsin-BPTI interface that seem to destabilise the complex by it adopting a less optimized packing and by tilting the BPTI molecule up to 15  compared to the native trypsin-BPTI complex. # 2000 Academic Press Keywords: bovine pancreatic trypsin inhibitor; serine proteinase speci®city; trypsin; chymotrypsin; plasmin *Corresponding author Introduction The interaction between canonical (also called ``standard mechanism'' or ``small'') protein inhibi- tors of serine proteinases and their cognate enzymes is one of the most extensively studied models of protein-protein recognition (Janin & Chothia, 1990; Jones & Thornton, 1996; Otlewski et al., 1999). In the complex, about 10 to 17 amino acid residues on the inhibitor site and 17 to 29 resi- dues of the proteinase make numerous van der Waals and hydrogen bond interactions. The princi- pal contact area on the inhibitor side is formed through a sequential epitope called the proteinase binding loop spanning from position P 3 to P 3 0 (notation by Schechter & Berger, 1967). The loop is convex and exhibits an extended conformation, which signi®cantly protrudes from the protein scaffold and serves as a simple recognition motif. The central section of the loop contains the solvent exposed P 1 -P 1 0 peptide bond, called the reactive site, which can be cleaved by a serine pro- teinase. The fully exposed side-chain of position P 1 is of particular importance for the proteinase- inhibitor association energy and, indeed, typical trypsin inhibitors have Arg/Lys at P 1 , whereas chymotrypsin inhibitors have Leu or Met. In many inhibitor families the amino acid at the P 1 position is recognized by a high degree of variability, e.g. E-mail address of the corresponding author: otlewski@bf.uni.wroc.pl Abbreviations used: BPTI, bovine pancreatic trypsin inhibitor; APPI, amyloid b-protein precursor inhibitor domain; TAP, tick anticoagulant peptide; TF-FVIIa, the complex between tissue factor and active factor VIIa; TFPI, tissue factor proteinase inhibitor. DSC, differential scanning calorimetry; IPTG, isopropyl-b-D- thiogalactopyranoside; K a , association constant; T den , temperature of denaturation; ÁH cal , calorimetric enthalpy change; ÁH vH , van't Hoff enthalpy change; ÁC p, den , denaturation heat capacity change; ÁG a , association free energy change; ÁG den , unfolding free energy change; k cat , catalytic constant; K m , Michaelis constant; NCS, non-crystallographic symmetry. doi:10.1006/jmbi.2000.3935 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 301, 205±217 0022-2836/00/010205±13 $35.00/0 # 2000 Academic Press