Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays

Abstract

Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation.

Engineering an Osmosensor by Pivotal Histidine Positioning within Disordered Helices

“…These studies challenged the common notion of a unidirectional relay that coupled sensing by extracytoplasmic domains of a receptor to the cytoplasmic functional site through large-scale conformational changes in the transmembrane segments (Chervitz and Falke, 1996;Gushchin et al, 2017;Hall et al, 2011;Hulko et al, 2006;Kitanovic et al, 2011;Ottemann et al, 1999). Indeed, transmembrane signaling is also regulated via phospholipid interactions with transmembrane and cytoplasmic domains (daCosta et al, 2009;Dowhan, 1997;Ghosh et al, 2017;Lee, 2003Lee, , 2004Leonard and Hurley, 2011;Phillips et al, 2009;Tsaloglou et al, 2011).…”

“…This observation highlighted the modularity of HKs and the underrecognized twoway allostery whereby stimulus sensing is achieved by generating cytoplasmic signals from extracellular stimuli (Figure 1A). Membrane-anchored HKs are also regulated by integral and peripheral lipid interactions (Ghosh et al, 2017). These studies challenged the common notion of a unidirectional relay that coupled sensing by extracytoplasmic domains of a receptor to the cytoplasmic functional site through large-scale conformational changes in the transmembrane segments (Chervitz and Falke, 1996;Gushchin et al, 2017;Hall et al, 2011;Hulko et al, 2006;Kitanovic et al, 2011;Ottemann et al, 1999).…”

A Modular High-Throughput In Vivo Screening Platform Based on Chimeric Bacterial Receptors

“…[28][29] An additional advantage to this system is that the cytoplasmic domain of EnvZ (EnvZc) has been previously shown to be sufficient for osmosensing. [30][31][32] Therefore, we can easily determine whether the osmosensing functionality can be retained in chimeras possessing a periplasmic domain that sense a stimulus not directly related to osmosensing.…”

The role of polyproline motifs in the histidine kinase EnvZ

“…The C-terminal cytoplasmic part of EnvZ comprises three distinct domains, HAMP, HisKA and HATPase [ 29 , 36 ]. Although the periplasmic domain is assumed to be involved in stimulus perception, recent studies show that the cytoplasmic portion can sense intracellular alterations resulting from extracellular osmotic changes [ 31 – 33 , 37 ]. EnvZ is bifunctional, acting as a kinase under conditions of high external osmolarity, and as a phosphatase at low osmotic pressures [ 38 ].…”