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Protein Science (2004), 13:325-331. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Disabling the folding catalyst is the last critical step in {alpha}-lytic protease folding

Erin L. Cunningham1 and David A. Agard2,3

1 Graduate Group in Biophysics,
2 Howard Hughes Medical Institute, and
3 Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California 94143-2240, USA

(RECEIVED August 21, 2003; FINAL REVISION October 15, 2003; ACCEPTED October 16, 2003)



Abstract

Alpha-Lytic protease ({alpha}LP) is an extracellular bacterial pro-protease marked by extraordinary conformational rigidity and a highly cooperative barrier to unfolding. Although these properties successfully limit its proteolytic destruction, thereby extending the functional lifetime of the protease, they come at the expense of foldability (t1/2 = 1800 yr) and thermodynamic stability (native {alpha}LP is less stable than the unfolded species). Efficient folding has required the coevolution of a large N-terminal pro region (Pro) that rapidly catalyzes {alpha}LP folding (t1/2 = 23 sec) and shifts the thermodynamic equilibrium in favor of folded protease through tight native-state binding. Release of active {alpha}LP from this stabilizing, but strongly inhibitory, complex requires the proteolytic destruction of Pro. {alpha}LP is capable of initiating Pro degradation via cleavage of a flexible loop within the Pro C-terminal domain. This single cleavage event abolishes Pro catalysis while maintaining strong native-state binding. Thus, the loop acts as an Achilles’ heel by which the Pro foldase machinery can be safely dismantled, preventing Pro-catalyzed unfolding, without compromising {alpha}LP native-state stability. Once the loop is cleaved, Pro is rapidly degraded, releasing active {alpha}LP.

Keywords: {alpha}-Lytic protease; protein folding; pro region; secondary cleavage; degradation

Abbreviations: {alpha}LP, {alpha}-lytic protease • Pro, wild-type pro region • Int, the intermediate state of {alpha}LP • Nat, the native state of {alpha}LP • Pro•Nat, complex of Pro with native of {alpha}LP • TEV, tobacco etch virus protease • ProTEVloop, Pro with the disordered loop replaced with a TEV protease recognition site • CD, circular dichroism

Reprint requests to: David A. Agard, Howard Hughes Medical Institute and the Department of Biochemistry and Biophysics, University of California at San Francisco, 600 16th Street, Room S412, San Francisco, CA 94143-2240, USA; e-mail: agard{at}msg.ucsf.edu; fax: (415) 476-1902.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03389704.


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