The N-end rule degradation pathway : substrate recognition and staged delivery to the CIpAP protease by the CIpS adaptor protein
Author(s)
Román Hernández, Giselle![Thumbnail](/bitstream/handle/1721.1/65296/745057395-MIT.pdf.jpg?sequence=5&isAllowed=y)
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Alternative title
Substrate recognition and staged delivery to the CIpAP protease by the CIpS adaptor protein
Other Contributors
Massachusetts Institute of Technology. Dept. of Biology.
Advisor
Tania A. Baker.
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Regulated protein degradation is crucial in the regulation of many physiological processes as well as in protein quality control. In all organisms, ranging from bacteria to mammals, ATP-dependent proteases carry out regulated protein degradation in order to maintain homeostasis as well as to respond to stress. ATP-dependent proteases are responsible for the degradation of a broad array of substrates and for that reason, a high degree of substrate specificity is required in order to target only desired proteins for destruction. Adaptor proteins can provide ATP-dependent proteases an extra-layer of specificity by binding and delivering a specific class of substrates, therefore regulating the activity of the protease. Understanding how adaptor proteins work in combination with their partner protease will provide a better understanding on how specificity is achieved by these proteolytic machines. In this thesis, different aspects of the bacterial N-end rule degradation pathway are examined. The N-end rule is a highly conserved degradation pathway that relates protein stability to the identity of its N-terminal residue in both prokaryotes and eukaryotes. For example, in bacteria, Tyr, Phe, Trp, and Leu serve as degradation signals when located at the N-terminus of a protein. In E. coli, the bacterial adaptor CIpS recognizes these signals and delivers these substrates to the AAA+ protease CIpAP. Here, we present the first crystal structure of a bacterial N-end rule adaptor, CIpS, bound to a peptide mimic of an N-end rule substrate. This structure pioneered the understanding behind the basis of N-end rule recognition by CIpS. The CIpS structure reveals the adaptor recognizes the peptide a-amino group via hydrogen bonding and shows that the peptide's N-terminal side chain is buried in a deep hydrophobic cleft that preexists on the surface of CIpS. We also present here the crystal structures of CIpS alone and engaged with peptides containing the rest of the primary N-end degrons (N-terminal phenylalanine, leucine, and tryptophan). These structures, together with the first structure of CIpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule residues. Moreover, we show that mutation of critical CIpS contact residues impairs substrate delivery to and degradation by the AAA+ protease CIpAP. In addition to the structural studies, the biochemical studies presented here provide a better understanding on how CIpS and CIpA work together for efficient N-end rule substrate delivery. Here, we show that substrate binding is enhanced substantially when CIpS binds ClpA6. Reciprocally, N-end-rule substrates increase CIpS affinity for CIpA6. Some of the features required for enhanced binding include the substrate N-end residue and the substrate first peptide bond. It also requires multiple features of CIpS, including a side chain that contacts the substrate a-amino group and two regions of a flexible N-terminal extension (NTE). We also show that enhancement in affinity requires the N domain and AAA+ rings of CIpA to be connected by a sufficiently long linker. One major novel finding uncovered in this thesis is that the CIpS NTE can be engaged by the CIpA translocation pore, but CIpS resists unfolding/degradation. We propose a staged-delivery model that illustrates how intimate contacts between the substrate, adaptor, and protease reprogram specificity and coordinate handoff from the adaptor to the CIpAP proteolytic machine.
Description
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2011. "June 2011." Cataloged from PDF version of thesis. Includes bibliographical references (p. 158-165).
Date issued
2011Department
Massachusetts Institute of Technology. Department of BiologyPublisher
Massachusetts Institute of Technology
Keywords
Biology.