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Published on: March 18, 2010
Structure of Escherichia coli aminopeptidase P in complex with the inhibitor apstatin
Stephen C Graham1, Megan J Maher, William H Simmons
1School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia.
Abstract:
Aminopeptidase P (APPro) is a metalloprotease whose active site includes a dinuclear manganese(II) cluster. The enzyme cleaves the N-terminal residue from a polypeptide when the second residue is proline. A complex of Escherichia coli APPro (EcAPPro) with an inhibitor, apstatin [N-(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl-L-prolyl-L-prolyl-L-alaninamide], has been crystallized. Apstatin binds to the active site of EcAPPro with its N-terminal amino group coordinated to one of the two Mn(II) atoms at the metal centre. The apstatin hydroxyl group replaces a hydroxide ion which bridges the two metal atoms in the native enzyme. The first proline residue of apstatin lies in a small hydrophobic cleft. The structure of the apstatin-EcAPPro complex has been refined at 2.3 A resolution with residuals R = 0.179 and R(free) = 0.204. The structure of the complex illustrates how apstatin inhibits APPro and suggests how substrates may bind to the enzyme, but the basis of the proline-specificity remains elusive.
Insights
Aminopeptidase P (APPro) is a metalloprotease that cleaves specific protein bonds. Crystallization of its complex with the inhibitor apstatin reveals binding details, aiding in understanding enzyme inhibition.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Aminopeptidase P (APPro) is a metalloprotease crucial for cleaving N-terminal residues when the second residue is proline.
- Its active site features a dinuclear manganese(II) cluster, essential for catalytic activity.
Purpose of the Study:
- To elucidate the structural basis of Aminopeptidase P inhibition by apstatin.
- To understand the binding interactions within the active site of Escherichia coli APPro (EcAPPro).
Main Methods:
- Crystallization of the EcAPPro-apstatin complex.
- X-ray diffraction analysis and structural refinement at 2.3 Å resolution.
- Analysis of atomic coordinates and interactions within the enzyme-inhibitor complex.
Main Results:
- The crystal structure of the EcAPPro-apstatin complex was determined with R = 0.179 and R(free) = 0.204.
- Apstatin binds to the active site, with its N-terminal amino group coordinating to a manganese ion and its hydroxyl group replacing a bridging hydroxide.
- The first proline of apstatin occupies a hydrophobic cleft, providing insights into inhibitor binding.
Conclusions:
- The determined structure illustrates the mechanism of apstatin inhibition of APPro.
- The findings suggest potential substrate binding modes for APPro.
- The structural data, however, do not fully explain the enzyme's proline-specificity.
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