2.3. Interfaces revealed by a chemical crosslinker coupled to
MS
A bifunctional crosslinker with a specific reactivity towards amino acid
residues or naturally occurring bonds in biomolecules can be usefully
employed to ‘freeze’ a protein complex in the solution phase. The
technique avoids the need for mutagenesis or derivatization of
interacting molecules which sometimes interferes with a natural binding
mode of interaction, and necessitates integrative analyses in tandem
with mass spectrometry due to rich but complex dataset generated upon
crosslinking. A chemical crosslinker is generally applied to a purified
biomolecular complex to eventually obtain comprehensive distance
information between residues or bonds thereof (Figure 2B). The
intrinsically disordered domain (IDD) of a protein has little propensity
to form a fixed or ordered conformation. Whereas some IDDs are involved
in functional molecular interaction, their flexible structure or
transient binding behavior often limits the investigation of binding
interfaces by conventional spectroscopic methods. A chemical linker
approach has proven effective to address this issue. The tumor
suppressor p53 which is known for its inherent structural flexibility
and resistance to crystallization was subjected to the chemical
crosslinking using an amine-reactive bifunctional linker coupled to mass
spectrometry. The analyses uncovered novel information regarding the
dimer-dimer interface of the p53 tetramer as well as a large degree of
flexibility of the C-terminal domain and DNA-dependent conformational
changes (Arlt et al., 2017; Arlt et al., 2015).
Moreover, amine-specific linkers with different reactive groups and
spacer distances were utilized to dissect the flexible or disordered
interfaces of macromolecular complexes such as MICAL3, ELKS, and Rab8A;
SLAIN2, CLASP2, and ch-TOG (Liu et al., 2017); NLRP1 and DPP9
(Hollingsworth et al., 2021); Rpn2 and Rpn13 (Gong et al., 2020). Recent
developments in linker chemistry have produced crosslinker reagents that
are cleavable, photo-activatable, or heterobifunctional, permitting
controlled crosslink reactions, low background, improved sensitivity
(Beard et al., 2021; Krist and Statsyuk 2015), and even in a native
cellular environment (Xie et al., 2021). For instance, a crosslinker
bearing a photoreactive moiety as well as a reducible spacer have been
developed. A bait protein labeled with the crosslinker at a defined
residue is photo-crosslinked to a prey protein after which a reductive
removal of a bait protein leaves a thiol-containing fragment of the
crosslinker onto the target that can be located by MS (Horne et al.,
2018; Mintseris and Gygi 2020). This technique improves MS sensitivity
by eliminating the background from bait peptides and, depending on the
site of crosslinker labeling and spacer length, would provide structural
information surrounding the interfacial region.
A substantial amount of interfacial information attained in these
biological environments was completely new or contradictory to
information previously obtained from the crystallographic or cryo-EM
structures, enabling remodeling or refinement by the combinatorial data
processing. The technique also successfully probed interfacial residues
responsible for protein oligomerization, which had been poorly resolved
by crystallography (Karagöz et al., 2017). Abovementioned and related
studies are summarized in the header rows of ‘photo-crosslinker’ and
‘chemical crosslinker’ (Table 1).