Identifying the requirement and mechanism of the dynamic yet kinetically stable ClpXP interface

AAA+ (ATPases Associated with various cellular Activities) proteases carry out regulated degradation and protein quality control. To achieve these goals, a barrel-shaped peptidase, with active sites in a sequestered chamber, cooperates with an attached AAA+ hexamer that recognizes, unfolds and translocates the protein substrate into the degradation chamber. The interaction between the AAA+ hexamer and peptidase must be specific and stable enough to ensure efficient degradation. However, information about the assembly of these proteases is limited. The subject of this thesis is the AAA+ ClpXP protease from Escherichia coli. ClpX is a AAA+ hexamer, and ClpP is a self-compartmentalized peptidase. Previous experiments have shown that ATP is required for assembly of active ClpXP complexes. Moreover, the IGF loops of ClpX are known to be important for stabilizing assembly. In Chapter 2, I use bio-layer interferometry (BLI) to determine the rates of ClpXP assembly and disassembly under different nucleotide conditions. ATP or ATP[gamma]S, a slowly hydrolyzed derivative, must occupy at least a subset of ClpX subunits allow ClpP binding. Moreover, I find that ClpX can only dissociate from ClpP once bound ATP is hydrolyzed to ADP and inorganic phosphate. As long as ClpX is ATP-bound, the ClpXP complex remains kinetically stable for more than three hours. However, even in the presence of ATP, the complex dissociates rapidly when ADEP, a small-molecule mimic of the IGF loop, is added. These results imply that the ClpXP interface is highly dynamic, with individual IGF-loops constantly unbinding and rebinding ClpP. In Chapter 3, I probe why ATP is important for ClpP binding and interrogate the importance of the sequence, length, and number of IGF-loops in ClpP binding. I found that ATP/ATP[gamma]S does not change the exposure of the IGF-loops but rather their proximity. At least four loops are required for stable binding, the length of the IGF loop is important, and residues in addition to the actual IGF sequence also play a role in complex stability. Finally, I discovered that a full complement of IGF loops is important both to allow rapid degradation and for degradation processivity because mutants with less than six loops do not appear to fully open the ClpP pore.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2017

Author dc:creator

• Amor, Alvaro Jorge

Advisor dc:contributor.advisor

• Robert T. Sauer.

• Biology.

Statement dc:rights

• MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written 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/1721.1/112890

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/112890