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.