1. Introduction
Viability of all forms of life is crucially reliant on homeostasis of
protein interactome. Therefore, elucidation of networks involving
protein-protein or protein-ligand interactions is a cornerstone for
understanding the complexity of biological processes. Additionally,
comprehensive informatics of protein interactions serves as a useful
template for drug discovery and drug repositioning (Payandeh and Volgraf
2021; Scott et al., 2016; Wang et al., 2021). A multitude of methods
have been developed to study the protein interactome with high
throughput and accuracy (Rao et al., 2014). While the protein
interactome illustrates the global network and relation of proteins and
ligands, mechanistic insight at molecular level into each set of protein
interactions further requires high-resolution technologies capable of
probing an array of critical motifs and residues, defined herein as an
interface, that are collectively responsible for accurate molecular
recognition and energetic stabilization.
Elementary bioanalytic methods readily available in most molecular
biology laboratories are size exclusion chromatography (SEC) and native
PAGE which examine if biomolecules under investigation do recognize each
other, albeit with little information of binding interfaces. These
methods macroscopically detect a change in mobility of biomolecules
through a matrix, attributed by size increase and charge variation upon
molecular association. Quantitative measurements of kinetics and
thermodynamics parameters involved in molecular interactions require
real-time techniques. Surface plasmon resonance (SPR) and bio-layer
interferometry (BLI) employ sophisticated optical biosensors that
monitor minute changes of resonance signals, or light interference
quantitatively dependent upon intermolecular binding events (Dysinger
and King 2012; Rich and Myszka 2011). Data processing from repetitive
measurements in different concentrations of an analyte yields the rates
of association and dissociations as well as the strength of the
interaction in terms of affinity (KD). Microscale
thermophoresis (MST) technique is quick to derive KDfrom variations of a molecular movement of a capillary-filled,
fluorescently labeled ligand throughout microthermal gradient, affected
by its association with an analyte and subsequent changes in physical
properties (Seidel et al., 2013). Isothermal titration calorimetry (ITC)
is a unique method that investigates the interaction between
biomolecules without labeling or immobilization (Pierce et al., 1999).
By measuring differential changes in the heat supplied to the sample
cell, as compared to the blank cell, in which two binding partners are
incrementally mixed, ITC provides useful parameters related to the
bimolecular interaction including binding stoichiometry, enthalpy and
entropy changes as well as affinity. Each quantitative method has its
advantages, and might serve as a standard technique depending on the
amount, purity, and physicochemical properties of biomolecules under
investigation. Despite being more informative than SEC and PAGE, these
methods are still far from defining the binding interface which is
mainly responsible for various interaction parameters obtained in the
measurements.
Structural information of a binding interface at atomic resolution can
be derived by implementing X-ray crystallography, nuclear magnetic
resonance spectroscopy (NMR), or cryo-electron microscopy (Cryo-EM).
While X-ray crystallography is the most predominant in accessibility and
practice thus far, the size of the crystallographic interface is usually
smaller and the interface water content is higher in general compared to
the interface under native environment, referred to herein as
‘biological interface’, due to non-physiological but
crystallization-favored conditions inflicted on a protein complex
(Schreiber 2020). Recent advances of NMR and Cryo-EM in instrumentation
and software reliably extract structural information of protein
complexes in pseudo-physiological conditions which can then be processed
to reconstruct preexisting interfacial crystal contacts into a
near-native state or even newly visualize the binding interfaces not
resolved in X-ray crystallography. Cyro-EM and NMR provide unique and
additional advantages of facile sample preparation and delivery of
structural dynamics data, respectively. Still, variable and dynamic
natures of binding interfaces embedded in protein complexes complicates
the interpretation of spectroscopic data with reasonable resolution and
fidelity, rendering mechanistic details of many protein interactions as
yet to be explored.
Aside from the high resolution, another critical attribute expected from
structural information of protein complexes is the high fidelity which
presents the most probable and representative snapshot of biological
interfaces under in-solution native environment, contrasting to a static
condition in which X-ray crystallography and electron microscopy are
generally best suited. Despite high resolution provided by static
methods, however, a static interface features non-physiological water
content and accessibility due to non-specificity caused by invasive
conditions such as a wide range of pH, high ion strength, high protein
concentration, all of which are detrimental to obtaining high-fidelity
interfacial landscape. In addition to such empirical artifacts, a static
interface often loses fidelity due to inherent natures of certain
protein interactions which could be dynamic, transient, weak, or
mediated by an intrinsically disordered domain that adopts no static
conformation. Thus, in combination of X-ray crystallography and electron
microscopy, supplementary methods to obtain structural data from the
native solution-phase are of great necessity to probe a biological
interface with both high resolution and fidelity.
Chemical crosslinking (CLMS) and hydrogen-deuterium exchange (HDMS),
both of which are coupled to mass spectrometry, have become unique and
complementary analytic methods to address abovementioned unmet needs in
structural biology. CLMS and HDMS are particularly valuable for
unveiling hot spots in interfaces that otherwise would be cryptic or of
poor fidelity, thus enabling the construction of a new interfacial
landscape of an unknown protein complex or reconstitution of a
preexisting protein interface, and subsequently aid in the
structure-based design of novel modulators of binding interfaces with a
high success rate (Arkin et al., 2014; Chen et al., 2012). In this
review, we briefly describe how CLMS and HDMS map the interfaces of
protein complexes with high fidelity and their integration with other
structural information to yield native-like high-order structures
assembled via biological interfaces. We then discuss recent efforts that
successfully employed CLMS and/or HDMS to reveal dynamic, transient, or
intrinsically disordered interfaces found in various protein complexes
that had been challenging to probe via X-ray crystallography and
electron microscopy.
Mass spectrometry-coupled techniques, CLMS and HDMS, provide novel means
of landscaping a biological interface. In contrast to abovementioned
spectroscopic techniques focusing on electronic or electromagnetic
properties of respective atoms, CLMS and HDMS delve into reciprocal
behavior of residues with other residues or matters surrounding them
under a native condition. The resultant mass changes are detected to
calculate corresponding inter-residue distances or solvent
accessibility. Although structural information obtained from CLMS and
HDMS is generally intermediate in resolution, it provides improved
fidelity by repositioning known residues in a static interface or
identifying unknown residues residing in a dynamic, transient, or
disordered interfacial region.