Tuning strength and specificity in the N-end rule

Abstract

Protein degradation is a central mechanism in the regulation of gene expression and activity. Proteolysis regulates not only homeostatic activities, but also the cell's responses to stress. A recurring question underlying this regulatory process is the specificity of substrate selection by the proteolytic machinery. I designed an unbiased selection to isolate N-terminal degradation sequences in vivo, which led to a collection of N-end rule signals. The N-end rule describes how the identity of a protein's N-terminal residue determines its metabolic stability. In E. coli, CIpAP is the principal protease that degrades proteins bearing an N-terminal phenylalanine, tyrosine, tryptophan, or leucine residue. The CIpS adaptor, which displays homology to eukaryotic ubiquitin ligases that recognize N-end signals, is a recently discovered component of the bacterial N-end rule. Using the collection of N-end signals, I was able to demonstrate that ClpS enhances N-end degradation by ClpAP but is not required in vivo or in vitro. The collection of N-end signals also provided insight into the role of sequence context in the N-end rule. Specifically, acidic residues and the length of the N-end signal affect degradation rates in vitro. These defective N-end signals also allowed us to separately define recognition specificities of ClpS and ClpAP. Whereas ClpS bound poorly to acidic N-end signals, CIpAP was unable to degrade substrates with short N-end sequences. Although two decades of biochemical and cellular data support the importance of the Nterminal residue in N-end degradation, there has been no structural information explaining how a single residue is recognized as a degradation signal.

(cont.) To this end, we solved a cocrystal structure of CIpS in complex with an N-end peptide. CIpS uses an extensive hydrogen bonding network to dock the a-amino group and a cavity lined with hydrophobic residues to recognize the N-terminal residue. Furthermore, mutation of the hydrophobic cavity altered the specificity of CIpS toward N-terminal residues. Together these findings attribute molecular functions to CIpS and ClpAP in the bacterial N-end rule and define sequence rules for the N-end signal. Furthermore, this work provides the tools and background for investigating the mechanism of substrate delivery by ClpS to ClpAP.