Membrane Anchoring Amplifies Osmosensing by the Inner Membrane Receptor EnvZ

Abstract

A fundamental question in signalling biology is how do receptors transmit the effects of extracellular stimuli across membranes? Transmembrane segments that connect a receptor extracellular domain to its intracellular domain are often considered to be critical elements for transmitting these signals. The dynamics of receptor signalling is not clearly understood, as there are few biophysical probes to directly monitor transmembrane regions. Amide Deuterium Exchange Mass Spectrometry (HDXMS) provides an excellent method to describe the conformational dynamics of membrane bound proteins. Our recently published studies with the cytoplasmic domain of the bacterial inner membrane osmosensor kinase EnvZ (EnvZc) have revealed the molecular basis for osmosensing through modulation of helix-coil transitions in a critical four helix bundle subdomain (Wang, LC, Morgan, L, Godakumbura, P, Kenney, LJ and Anand, GS EMBO J. 31(11):2648-59, 2012). This study highlighted the importance of local folding-unfolding equilibria in the functioning of EnvZ signalling. Most surprising was that a cytoplasmic deletion fragment of EnvZ lacking the transmembrane helices was able to fully rescue the osmosensing function in a strain lacking the envZ gene. This immediately raised the question as to what is the function of membrane anchoring? Our preliminary results indicate that the helix-coil transitions and associated local unfolding we observed in EnvZc also occur in transmembrane helix regions in EnvZ. Conformational dynamics of full-length EnvZ embedded within nanodiscs indicate that the osmosensing core of EnvZ shows greater deuterium exchange compared to EnvZc, suggesting a regulatory role of the membrane in modulating the autokinase activity of EnvZ. Our results reveal that membrane anchoring is responsible for ‘damping down' the helix-coil transitions and likely increases the sensitivity range for the receptor. These results are broadly relevant to all receptors. Supported by Mechanobiology RCE and VA-1IOBX000372 to LJK.