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Protein Science (2004), 13:1056-1070. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Trypsin specificity as elucidated by LIE calculations, X-ray structures, and association constant measurements

Hanna-Kirsti Schrøder Leiros1,4, Bjørn Olav Brandsdal1,2, Ole Andreas Andersen1,2, Vibeke Os1,2, Ingar Leiros1,4, Ronny Helland1,2, Jacek Otlewski5, Nils Peder Willassen3 and Arne O. Smalås1,2

1 Protein Crystallography Group, Department of Chemistry, Faculty of Science,
2 The Norwegian Structural Biology Centre, and
3 Institute of Medical Biology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway
4 The European Synchrotron Radiation Facility (ESRF), F-38043 Grenoble Cedex, France
5 Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50–137 Wroclaw, Poland

(RECEIVED October 29, 2003; FINAL REVISION December 8, 2003; ACCEPTED December 8, 2003)



Abstract

The variation in inhibitor specificity for five different amine inhibitors bound to CST, BT, and the cold-adapted AST has been studied by use of association constant measurements, structural analysis of high-resolution crystal structures, and the LIE method. Experimental data show that AST binds the 1BZA and 2BEA inhibitors 0.8 and 0.5 kcal/mole more strongly than BT. However, structural interactions and orientations of the inhibitors within the S1 site have been found to be virtually identical in the three enzymes studied. For example, the four water molecules in the inhibitor-free structures of AST and BT are channeled into similar positions in the S1 site, and the nitrogen atom(s) of the inhibitors are found in two cationic binding sites denoted Position1 and Position2. The hydrophobic binding contributions for all five inhibitors, estimated by the LIE calculations, are also in the same order (-2.1 ± 0.2 kcal/mole) for all three enzymes. Our hypothesis is therefore that the observed variation in inhibitor binding arises from different electrostatic interactions originating from residues outside the S1 site. This is well illustrated by AST, in which Asp 150 and Glu 221B, despite some distance from the S1 binding site, lower the electrostatic potential of the S1 site and thus enhance substrate binding. Because the trends in the experimentally determined binding energies were reproduced by the LIE calculations after adding the contribution from long-range interactions, we find this method very suitable for rational studies of protein–substrate interactions.

Abbreviations: AST, anionic salmon trypsin • CST, cationic salmon trypsin • BT, bovine trypsin • CHST, chum salmon trypsin • 1BZA, benzamidine • 2BEA, benzylamine • ANL, aniline • AMC, aminomethylcyclohexane • FBA, 4-fluorobenzyl • 3PEA, phenylethylamine • 4PPA, phenylpropylamine • 5PBA, phenylbutylamine • BPTI, bovine pancreatic trypsin inhibitor • AST-BPTI, AST complexed with BPTI (1BZX) • BT-1BZA, BT complexed with benzamidine (3PTB) • BT-FBA, BT with 4-fluorobenzylamine (1TNH) • BT-AMC, BT with amino-methylcyclohexane (1TNG) • BT-3PEA, BT with phenylethylamine (1TNJ) • BT-4PPA, BT with phenylpropylamine (1TNK) • BT-5PBA, BT with phenylbutylamine (1TNI) • BT-K15G, BT complexed with BPTI and P1 Gly (3BTG) • BT-K15F, BT complexed with BPTI and P1 Phe (3BTP) • LIE, linear interaction energy • MPD, 2-methyl-2,4-pentanediol • GOL, glycerol • MD, molecular dynamics

Keywords: trypsin; inhibitor specificity; electrostatic interactions; cold-adaptation; molecular dynamics; binding free energy

Reprint requests to: Arne O. Smalås, University of Tromsø, N-9037 Tromsø, Norway; e-mail: arne.smalas{at}chem.uit.no; fax: 47-776-44737.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03498604.


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