Physiological N-end rule substrates in E. coli and the molecular details of their ClpS-mediated degradation by ClpAP

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry, School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The N-end rule is a highly conserved, hierarchical protein degradation pathway that relates the half-life of a protein to its N-terminal residue. Although the physiological role of this pathway remains unclear in E. coli, many of the components of the pathway are well defined. Proteins degraded by this pathway contain an N-terminal recognition motif (referred to as an N-degron) that is composed of a primary destabilising residue (Leu, Phe, Trp or Tyr) and an unstructured region containing a hydrophobic motif. In general, N-degron bearing substrates are thought to be generated by an endoproteolytic cleavage. In some cases, the cleaved protein may also be modified by the enzyme LFTR in a non-ribosomal manner. Regardless of their mode of generation, all N-degron bearing substrates are recognised by the adaptor protein (ClpS) and delivered to protease ClpAP, for degradation. To date, only two physiological, N-degron bearing substrates have been verified (i.e. PATase and Dps) one of which is modified by LFTR, in a non-conventional manner. In this study the substrate proteome of LFTR has been determined. From this proteome, one protein (AldB) was modified by an uncharacterised activity of LFTR. The new specificity of LFTR was confirmed in vitro, using a range of peptide and protein substrates. Importantly, this modification of AldB is essential for its ClpAPS-mediated turnover, in vitro. In addition, a systematic analysis of the LFTR specificity using a peptide array, revealed that positively charged residues in the penultimate position favour conjugation, while negatively charged residues disfavour conjugation. Finally, mutational studies and structural analysis of several putative physiological N-end rule substrates identified that only one class of substrates requires the hydrophobic motif for delivery to ClpAP. Proteins containing a N-terminal helix require the hydrophobic motif while proteins lacking an N-terminal helix use an alternative mechanism of delivery.