4. Probing biological interfaces by integrative approaches
While CLMS and HDMS may stand alone as the techniques to map the
biological interface, their integration aided by computational modeling
with the dataset obtained from X-ray crystallography, EM, or NMR can
significantly improve spatial resolution and coverage of binding
interfaces to provide comprehensive illustrations. For example, Zhang et
al. first implemented CLMS and HDMS together to extract previously
unknown information on the epitope and paratope interface of a
programmed cell death-1 (PD-1) and the corresponding antagonistic
antibody. In the next round of refinement, the suggested critical
binding residues and distance restraints were utilized to build
high-confidence binding models through molecular docking onto the
crystal structure (Zhang et al., 2020). While the epitope-paratope
relationships revealed by the approach were generally comparable with
those assigned by the crystal structure, one cryptic loop in PD-1 that
had not been crystallographically resolved due to its flexible nature
was found to be a non-epitope. The study demonstrated a complementary
role of CLMS and HDMS for accurate and detailed examination of a binding
interface. The same research group described a similar integrated
approach combining CLMS, HDMS, and molecular docking to probe the
binding interface of interleukin 7 (IL-7) complexed with its receptor
IL-7Rα. While the predicted model was generally in accordance with the
crystal structure, the approach newly discovered the C-terminal binding
region of IL-7, highlighting the value of integrative approaches to
obtain a high-confidence structural model (Zhang et al., 2019).
Interpretation of HDMS data using a predetermined crystal structure as a
template provides greater insight into reversible changes in regional
flexibility of binding interfaces. A junctional epitope antibody VHH6,
specifically recognizing a neo-epitope created only at the junction in
which IL-6 and gp80 are interlocked, was considered a molecular clamp as
shown in the ternary crystal structure. To understand the effect of VHH6
clamping on the structural flexibility of the junctional interface of
IL-6-gp80, Adams et al. compared the amounts of deuterium exchange
therein in the presence or absence of VHH6 (Adams et al., 2017). The
presence of VHH6 increased the rigidity in the local region spanning the
junction, stabilizing a transient interface between IL-6 and gp80.
Likewise, advances in CLMS techniques are better refining a
low-resolution interface solved by other methods into medium- to
high-resolution details. By applying multiple orthogonal crosslinking
chemistries to a target protein complex, Mintseris and Gygi could attain
a higher crosslinking density and improved sequence coverage (Mintseris
and Gygi 2020). Self-consistent analytic results could be mapped onto
cryo-EM models to define the interaction interface with high resolution
and confidence.
A dramatic contribution of HDMS in harmony with CLMS to modeling the
interface of an unstable macromolecular complex was presented by Shuka
et al (Shukla et al., 2014). While the human β2adrenergic receptor (β2AR) and β-arrestin-1 had crystal
structures reported individually, the β2AR-β-arrestin-1
complex was intolerant to experimental conditions in X-ray
crystallography or EM with only low-resolution map for the overall
conformation available. Remarkably, constraints provided by HDMS and
CLMS were mapped onto the preexisting data in an integrative manner,
resulting in the three-dimensional reconstruction of the complex that
displayed an unexpectedly extensive interface and a crucial involvement
of the finger loop in β-arrestin-1 in the complex formation. More
recently, such a combined approach investigated the mechanism of client
binding of the periplasmic chaperone SurA and identified dynamic
inter-domain interfaces that underwent substantial structural
reorganization in response to the substrate binding interface being
occupied by a client OmpX (Calabrese et al., 2020).