Structure-function relationship of serine protease-protein inhibitor interaction.

• Authors: Jacek Otlewski , Mariusz Jaskólski , Olga Buczek , Tomasz Cierpicki , Honorata Czapińska , Daniel Krowarsch , Arne O Smalas , Damian Stachowiak , Agnieszka Szpineta , Michał Dadlez
• Languages of publication: EN

Abstracts

EN

We report our progress in understanding the structure-function relationship of the interaction between protein inhibitors and several serine proteases. Recently, we have determined high resolution solution structures of two inhibitors Apis mellifera chymotrypsin inhibitor-1 (AMCI-I) and Linum usitatissimum trypsin inhibitor (LUTI) in the free state and an ultra high resolution X-ray structure of BPTI. All three inhibitors, despite totally different scaffolds, contain a solvent exposed loop of similar conformation which is highly complementary to the enzyme active site. Isothermal calorimetry data show that the interaction between wild type BPTI and chymotrypsin is entropy driven and that the enthalpy component opposes complex formation. Our research is focused on extensive mutagenesis of the four positions from the protease binding loop of BPTI: P1, P1', P3, and P4. We mutated these residues to different amino acids and the variants were characterized by determination of the association constants, stability parameters and crystal structures of protease-inhibitor complexes. Accommodation of the P1 residue in the S1 pocket of four proteases: chymotrypsin, trypsin, neutrophil elastase and cathepsin G was probed with 18 P1 variants. High resolution X-ray structures of ten complexes between bovine trypsin and P1 variants of BPTI have been determined and compared with the cognate P1 Lys side chain. Mutations of the wild type Ala16 (P1') to larger side chains always caused a drop of the association constant. According to the crystal structure of the Leu16 BPTI-trypsin complex, introduction of the larger residue at the P1' position leads to steric conflicts in the vicinity of the mutation. Finally, mutations at the P4 site allowed an improvement of the association with several serine proteases involved in blood clotting. Conversely, introduction of Ser, Val, and Phe in place of Gly12 (P4) had invariably a destabilizing effect on the complex with these proteases.

Keywords

EN

calorimetry; serine proteases; structural thermodynamics; protein inhibitor; protein-protein recognition

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2001
• Volume: 48
• Issue: 2
• Pages: 419-428

Dates

published

2001

received

2001-02-14

accepted

2001-04-30

Contributors

author

Jacek Otlewski

• Institute of Biochemistry and Molecular Biology, University of Wrocław, Wrocław, Poland

author

Mariusz Jaskólski

• Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland

author

Olga Buczek

• Institute of Biochemistry and Molecular Biology, University of Wrocław, Wrocław, Poland

author

Tomasz Cierpicki

• Institute of Biochemistry and Molecular Biology, University of Wrocław, Wrocław, Poland

author

Honorata Czapińska

• Institute of Biochemistry and Molecular Biology, University of Wrocław, Wrocław, Poland

author

Daniel Krowarsch

• Institute of Biochemistry and Molecular Biology, University of Wrocław, Wrocław, Poland

author

Arne O Smalas

• Protein Crystallography Group, Department of Chemistry, University of Tromsø, Tromsø, Norway

author

Damian Stachowiak

• Institute of Biochemistry and Molecular Biology, University of Wrocław, Wrocław, Poland

author

Agnieszka Szpineta

• Institute of Biochemistry and Molecular Biology, University of Wrocław, Wrocław, Poland

author

Michał Dadlez

• Institute of Physics, Department of Biophysics, Warsaw University, Warszawa, Poland

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