Journal of Biological Chemistry
Volume 285, Issue 18, 30 April 2010, Pages 13958-13965
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Protein Structure and Folding
Proenzyme Structure and Activation of Astacin Metallopeptidase*

https://doi.org/10.1074/jbc.M109.097436Get rights and content
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Proteolysis is regulated by inactive (latent) zymogens, with a prosegment preventing access of substrates to the active-site cleft of the enzyme. How latency is maintained often depends on the catalytic mechanism of the protease. For example, in several families of the metzincin metallopeptidases, a “cysteine switch” mechanism involves a conserved prosegment motif with a cysteine residue that coordinates the catalytic zinc ion. Another family of metzincins, the astacins, do not possess a cysteine switch, so latency is maintained by other means. We have solved the high resolution crystal structure of proastacin from the European crayfish, Astacus astacus. Its prosegment is the shortest structurally reported for a metallopeptidase, and it has a unique structure. It runs through the active-site cleft in reverse orientation to a genuine substrate. Moreover, a conserved aspartate, projected by a wide loop of the prosegment, coordinates the zinc ion instead of the catalytic solvent molecule found in the mature enzyme. Activation occurs through two-step limited proteolysis and entails major rearrangement of a flexible activation domain, which becomes rigid and creates the base of the substrate-binding cleft. Maturation also requires the newly formed N terminus to be precisely trimmed so that it can participate in a buried solvent-mediated hydrogen-bonding network, which includes an invariant active-site residue. We describe a novel mechanism for latency and activation, which shares some common features both with other metallopeptidases and with serine peptidases.

Crystal Structure
Enzyme Inactivation
Enzyme Processing
Enzyme Structure
Matrix Metalloproteinase
Metalloprotease
Peptidases
Protease
Metzincin
Proprotease

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This paper is dedicated to Wolfram Bode on the occasion of his 68th birthday and to acknowledge his groundbreaking contributions to the structural analysis of metallopeptidases.

The atomic coordinates and structure factors (code 3LQ0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

*

This work was supported by grants from European, Spanish, and Catalan public agencies (Strep FP7-223101-AntiPathoGN, BIO2008-04080-E, BIO2009-10334, CSD2006-00015, PSE-010000-2009-8, and 2009SGR1036) and by Deutsche Forschungsgemeinschaft Grant Sto185/3-3. Funding for data collection was provided by the European Synchrotron Radiation Facility.

1

Present address: The Overall Laboratory, Centre for Blood Research, University of British Columbia; Rm. 4.401, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.