Integrating hydrogen–deuterium exchange mass spectrometry with molecular dynamics simulations to probe lipid-modulated conformational changes in membrane proteins

Abstract

Biological membranes define the boundaries of cells and are composed primarily of phospholipids and membrane proteins. It has become increasingly evident that direct interactions of membrane proteins with their surrounding lipids play key roles in regulating both protein conformations and function. However, the exact nature and structural consequences of these interactions remain difficult to track at the molecular level. Here, we present a protocol that specifically addresses this challenge. First, hydrogen-deuterium exchange mass spectrometry (HDX-MS) of membrane proteins incorporated into nanodiscs of controlled lipid composition is used to obtain information on the lipid species that are involved in modulating the conformational changes in the membrane protein. Then molecular dynamics (MD) simulations in lipid bilayers are used to pinpoint likely lipid-protein interactions, which can be tested experimentally using HDX-MS. By bringing together the MD predictions with the conformational readouts from HDX-MS, we have uncovered key lipid-protein interactions implicated in stabilizing important functional conformations. This protocol can be applied to virtually any integral membrane protein amenable to classic biophysical studies and for which a near-atomic-resolution structure or homology model is available. This protocol takes~4 d to complete, excluding the time for data analysis and MD simulations, which depends on the size of the protein under investigation.

Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter

“…Rather, it reports on the stability of H-bond of the amide backbone which is mainly conditioned by two parameters: local structural dynamics and solvent accessibility [13,14]. We have shown previously that for a series of transporters, the changes in solvent accessibility can be correlated with conformational changes in most cases [15]. This is particularly helpful to understand the molecular mechanism of transporters as they switch between OF and IF conformations [16].…”

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

“…The unique ability of nanodiscs to be captured intact, coupled with their customizability, affords a broad range of biochemical applications and has expanded the repertoire of approaches that can be used to investigate peripheral membrane protein oligomerization and lipid interactions. For example, nanodiscs have been successfully integrated with HDX-MS [71,72]. In this context, the combination of nMS with top-down sequencing methods offers a chance to connect lipid binding with structural changes.…”

Structural predictions of the functions of membrane proteins from HDX-MS

“…Developments in HDX-MS methodologies have allowed the investigation of membrane protein dynamics under previously inaccessible conditions and environments. While detergent micelles remain the primary choice for membrane protein solubilisation, recent advances have enabled the interrogation of membrane protein dynamics in lipid-based environments, reminiscent of their natural conditions [22,26,32]. More specifically, researchers have used HDX-MS to probe the structural dynamics of membrane proteins embedded in SMALPs [26], nanodiscs [20,22] and proteoliposomes [21].…”

“…Continuing on membrane proteins, a recent protocol combined HDX-MS and MD simulations to investigate the role of lipids in modulating the conformational dynamics of membrane proteins from the Major facilitator superfamily of transporters, namely XylE, LacY and GlpT [22,32] (Figure 2). HDX-MS data was first collected for various membrane proteins reconstituted in lipid nanodiscs with tuneable compositions.…”

“…Although HDX is tolerant of lipids and other complex environments, interfacing lipids with the fast separation and digestions required for HDX-MS can lead to fouling of the liquid chromatography (LC) and digestion columns. These challenges can be mitigated through a number of workflow procedures including using a sawtooth LC method, running ‘clean blank’ injections, and pepsin washes after each lipid-containing sample to minimise the build-up of lipids [ 32 33 ]. Extensive details of how to manipulate the lipid environments surrounding membrane proteins for exploring lipid-mediated conformational changes can be found in a recently published protocol [ 32 ].…”