Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

Abstract

The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the ␣-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met 53 side chain directly enforces specificity by clashing with and excluding ␤-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.AAAϩ ATPase adaptor ͉ clpAP protease ͉ degradation tag ͉ degron

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

“…Short peptides with leucine or phenylalanine at the N terminus were effective inhibitors of LR-GFP Venus degradation, but the presence of a single hydrophilic residue at the N terminus of the peptides rendered them completely ineffective as inhibitors. These data are consistent with other studies showing that ClpS is needed to mediate degradation of proteins targeted by virtue of an N-degron (5,11,12,16).…”

“…ClpS binds a single molecule of N-end rule substrate (12,16). To test whether the efficiency of substrate delivery was dependent on the number of ClpS bound to ClpA 6 , we measured degradation as a function of LR-GFP Venus concentration with different stoichiometric ratios of ClpS to ClpA 6 .…”

“…Peptides with Phe, Tyr, Trp, or Leu at the N terminus bind to ClpS, and similar peptides with other N-terminal residues bind with substantially lower affinity (5). The crystal structure of ClpS with a bound peptide bearing an N-degron shows the hydrophobic side chain of the N-degron pointing into a deep hydrophobic pocket in ClpS and the ␣-amino group of the peptide forming an ion pair with an acidic residue on the rim of the hydrophobic pocket (12,16). Other contributions from hydrogen bonding between the main chain carbonyls and amides are limited and nonspecific.…”

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

“…The N-domain contains a hydrophobic binding pocket, whereas the UBR box uses several negatively charged side chains in binding pockets. [11][12][13] In a previous study, we confirmed that the L-conformation, protonated a-amine group, and hydrophobic side chains are essential N-degron structural components required for direct interaction with N-recognins. 14 Based on this conformational information, several small, biocompatible Arg/N-end rule inhibitors have been developed to target both the UBR box and the N-domain simultaneously.…”

The N-degradome of Escherichia coli

“…Another adapter, RssB, specifically targets the factor RpoS for degradation by ClpXP (14 -16). ClpA on the other hand interacts with the adapter ClpS, which binds proteins bearing one of a small subset of N-terminal residues and delivers them to ClpAP for degradation (17)(18)(19)(20)(21).…”