Specificity in protein-ligand recognition: characterisation of a novel conformational switch in the p62 UBA domain interaction with ubiquitin

The multi-functional p62 protein (also known as SQSTM1) regulates a diverse range of cellular processes involving in vivo interaction partners relevant to bone cell (osteoclast) and neuronal function. Mutations affecting p62 are a common cause of Paget's disease of bone (PDB), however, the precise disease mechanism in this skeletal disorder is unclear. Ubiquitin-associated (UBA) domains have been identified in many proteins as ubiquitin (Ub)-binding motifs; PDB mutations in p62 occur principally within its C-terminal UBA domain, with the implication that these pathological mutations may compromise interactions of the p62 protein with osteoclast protein targets already tagged with ubiquitin. More recently, mutations in other parts of the p62 sequence have been identified in PDB patients which still result in loss or impairment of ubiquitin-binding function. In structural studies of the UBA domain of p62 and its interaction with ubiquitin and poly-ubiquitin chains, we have recently demonstrated a novel structural re-organisation of the UBA upon binding ubiquitin that appears to provide an unique mechanism for specific regulation of ubiquitin-binding, given the participation of p62 in multiple signalling complexes. This binding mechanism suggests that PDB mutations in the UBA domain could affect ubiquitin binding specificity through both direct and indirect mechanisms that affect this conformational equilibrium. We plan to investigate the molecular origins of the interaction of much larger fragments of the p62 protein than simply the UBA domain, and investigate the underlying physiological basis for ubiquitin recognition with PDB mutations as valuable tools.

The p62/SQSTM1 protein acts as a scaffold protein in a number of signalling pathways that lead to NF?B activation. The C-terminal region of the protein (residues 387-436) has been identified as a ubiquitin-associated (UBA) domain that occurs in enzymes of the ubiquitin conjugation/deconjugation pathway. Mutations in the p62 protein cause Paget's disease of bone (PDB), a common disorder of the elderly characterised by excessive bone resorption and formation. The majority of PDB mutations to date are found in the UBA domain with the implication that these pathological mutations must compromise interactions of the p62 protein with a ubiquitinated target. In structural studies of the UBA domain of p62 and its interaction with ubiquitin and poly-ubiquitin chains, we have recently demonstrated a novel structural re-organisation of the UBA upon binding, that appears to provide an unique mechanism for specific regulation of ubiquitin-binding in the context of p62's participation in multiple signalling complexes. We plan to use naturally occurring PDB mutations to probe the mechanism of ubiquitin recognition by the p62 UBA domain, by examining how such mutations can affect the thermodynamics of the complexation process and the conformational equilibrium between the two states of the UBA domain, only one of which binds ubiquitin. We have shown that longer p62 constructs bind with higher affinity than the minimal UBA domain to monoubiquitin and that non-UBA domain mutations of p62 can perturb ubiquitin-binding, implicating non-UBA domain sequences in recognition and binding. We will examine the conformational switch mechanism in the context of longer p62 constructs, and use the PDB mutations to probe the critical determinants of ubiquitin recognition by the full-length p62 protein (1-440) and a variety of fragments. We have a range of biophysical methods available to us, in particular multi-nuclear NMR spectroscopy of 15N/13C-labelled proteins and mass spectrometry.