Regulation of ClpP : role of substrate gating and activation by ClpX

AAA+ self-compartmentalized proteases are an important class of proteome regulators that operate to selectively degrade protein substrates. All of these enzymes share the architectural theme of a hexameric ring unfoldase stacked axially onto a barrel-like peptidase, with six- or seven-fold symmetry and sequestered active sites. ClpXP is a model self-compartmentalized protease composed of the regulator ClpX and the serine protease ClpP. Proteolysis occurs by ClpX-dependent substrate selection, unfolding, and translocation into the degradation lumen of ClpP, where rapid and relatively non-specific peptide hydrolysis generates small peptide products. Prior work had shown that ClpP is unable to degrade polypeptides in the absence of ClpX, suggesting the existence of a mechanism that inhibits the activity of free ClpP. Structures of free ClpP show active sites geometrically competent to perform peptide-hydrolysis chemistry. However, some biochemical results suggested that N-terminal ClpP residues, which line the axial entrance pores, allosterically regulate these active sites. Through measurements of ClpP active-site reactivity, degradation of size-varied peptides, and mutagenesis of the N-termini, I found that peptide degradation is inhibited by steric occlusion, maintained by the N-terminal 3-stem loop and a-helix A of ClpP. The N-termini also participate in specifying substrate choice, as mutations within the axial channel prevent degradation of peptides containing stretches of charged amino acids. These data support a model in which ClpX binding opens the axial pore of ClpP to facilitate polypeptide translocation.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2010

Author dc:creator

• Lee, Mary Elizabeth, Ph. D. Massachusetts Institute of Technology

Advisor dc:contributor.advisor

• Robert T. Sauer.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/58374

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/58374