3.3. Protein-lipid interface
Membrane proteins are notoriously insusceptible to high-purity preparation for structural analyses due to their lipophilic nature and propensity to deformation when not in contact with membranes. Detergent micelles and vesicles are usually employed as standard formulations to isolate membrane proteins in a stable and functional state, thus allowing HDMS to probe conformational dynamics and molecular interactions of membranes proteins. Readers are referred to recent review articles that discussed the accomplishments of HDMS in structural biology of membrane proteins (Kaiser and Coin 2020; Martens and Politis 2020). Here we focus on the role of HDMS in exploring a heterogeneous interface between a membrane protein and phospholipids surrounding it. Hydrogen-deuterium exchange analysis is one of the least invasive methods that requires neither reactive chemicals nor harsh conditions, and thus readily operational under any preparative condition optimized for specific membrane proteins that behave in a reversible (peripheral protein) or permanent (integral protein) manner (Figure 3C). Moreover, neither high sample purity nor large sample quantity is essential for HDMS owing to recent advances in instrumentation and data processing software (Martens et al., 2019).
The power of HDMS, free of labeling or crosslinking, was well utilized in the study of transmembrane regions within an integral membrane protein. A GPCR model, β2-adrenergic receptor (β2AR) was prepared in micelles for HDMS analyses, and the overall HDMS profile showed that the transmembrane regions surrounded by lipids was much lower in deuterium exchange than the exposed regions, correlating well with the predicted seven-transmembrane structure of GPCRs (Duc et al., 2015). In another study, transporter proteins prepared in nanodiscs with different lipid compositions were analyzed by HDMS for changes in conformational equilibrium (Martens et al., 2018). It was found out that the charge-conserved, lipid-contacting interfaces of transporters were responsible for the conformational shift whose equilibrium was significantly affected by the lipid composition.
Tumor suppressor phosphatase and tensin homolog (PTEN) interacts with cell membranes in a switchable manner depending on dynamic conformational ensembles affected by phosphorylation (Jang et al., 2021). In addition to active dynamics, PTEN possesses intrinsically disordered tails at both termini which are considered important as membrane binding elements but not crystallizable. HDMS could unveil a novel mechanism of membrane interaction by PTEN, a behavior specifically driven by the membrane-binding interface at the N-terminal tail in which the extent of deuterium exchange was significantly dependent upon co-incubation of the lipid vesicles interacting with PTEN (Masson et al., 2016). Similarly, HDMS was employed to map the interface of sphingosine kinase 1 (SK1) and membrane vesicles. SK1 is an enzyme that catalyzes the conversion of sphingosine in membranes to sphingosine-1-phosphate (S1P). The study identified a positively charged motif on SK1 responsible for electrostatic interactions with membranes, and further demonstrated a contiguous interface, comprising an electrostatic site and a hydrophobic site, that interacted with membrane-associated anionic phospholipids (Pulkoski-Gross et al., 2018). More recently, a 390-kDa heterotetrameric lipid kinase Vps34 examined by HDMS has revealed its so-called ‘aromatic finger’ that interacts directly with lipid membranes and regulates the catalytic activity. A decreased rate of deuterium exchange in the finger region in the presence of lipid vesicles served as a signature for its defined role. Abovementioned and related studies are summarized in Table 2.