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).