The structural biology approach – from models to mechanism

Dr John Schwabe

Summer School 11-14 July 2006
The Møller Centre, Storeys Way, Cambridge, UK

The identification of mutations in regulatory proteins that result in human disease raises the fundamental question as to the molecular mechanisms that are perturbed so as to give rise to the disease state. In many cases a careful investigation of such mutants gives new insight into the normal biological functioning of the proteins in question.

This presentation concerns two mutations (V290M & P467L) in the nuclear receptor PPARg that are associated with a specific metabolic phenotype characterised by severe insulin resistance, early onset hypertension and partial lipodystrophy. Examination of the structure of the PPARg ligand binding domain shows that whilst the two mutations are distant in the primary sequence, they are in close proximity in the three dimensional structure in the vicinity of the C-terminal helix 12 of the receptor. These mutations suggest therefore that this is a critical region of the receptor for normal signaling. This fits well with previous studies, which suggested that helix 12 plays a critical role in regulating co-factor recruitment.

Based upon the human mutations, we hypothesized that the dynamic properties of helix 12 may be critical for the activation mechanism of PPARg and other nuclear receptors. Accordingly, we used fluorescence anisotropy techniques to directly monitor the mobility of helix 12 in PPARg. Our results suggest that helix 12 is significantly more mobile than the main body of the protein. Upon ligand binding helix 12 exhibits reduced mobility accounting for its role as a molecular switch. PPARg proteins containing the V290M and P467L mutations exhibit perturbations in the dynamic behavior of helix 12. These findings, prompted by the discovery of the human mutations, provided the first direct observations of the mobility of helix 12 and suggest that the dynamic properties of this helix are key to the regulation of transcriptional activity.

The opinions expressed in this paper are those of the speaker and do not necessarily reflect the views of the Society

Revised: 24-Aug-2006