2.2. Interfaces revealed by UAA incorporation and crosslinking coupled to MS
While the UAA-mediated photo-crosslinking technique facilitates a scan of interfacial residues on one side of the biomolecular complex, specific locations of the crosslinks made at the other binding molecules remain elusive. This is because photo-reactive UAA can be site-specifically incorporated in the target protein but its reactivity upon irradiation towards a binding partner protein is non-specific. Analytic improvements made through enzymatic fragmentation of the crosslinked complex followed by fingerprinting by high-resolution mass spectrometry opened up a new arena of probing two-sided binding interfaces of a protein complex with better resolution and fidelity (Figure 2B). For example, to investigate the mechanistic details of lipopolysaccharide transport through the integral membrane protein LptD/E complex, LptE was site-specifically modified with an UAA photo-crosslinker at various positions. The covalent intermolecular complexes LptD/E formed upon irradiation were observed in Western blotting and then subjected to tryptic digestion followed by high-resolution two-dimensional MALDI-TOF/TOF spectrometry. The UAA incorporated at the 150th position of LptE was found to be linked to S538 in LptD, specifically suggesting that a putative extracellular loop of the LptD β-barrel spanning a residue S538 was a major motif of interaction with LptE (Freinkman et al., 2011). In case a ligand is a small molecule that is amenable to derivatization with a photo-reactive group, the interface mapping can be performed without generating multiple protein mutants bearing a photo-reactive UAA. Diazirine-bearing cholesterol acting as a photoactivatable crosslinker as well as a cofactor for the glycine receptor was employed to investigate how the landscape encompassing contact residues between the cholesterol and the glycine receptor would vary depending on a cholesterol concentration. Crosslinks obtained after trypsin digestion were analyzed by the multidimensional MS to identify specific amino acids of the glycine receptor linked to the cholesterol with high sensitivity and subsequently map the cholesterol-protein interface altering as a function of cholesterol composition (Ferraro and Cascio 2018). A bifunctional UAA bearing a diazirine, for photo-crosslinking, and a terminal alkyne group, for bioorthogonal tagging, has been reported (Yang et al., 2020). A ligand harboring the bifunctional UAA captured a target protein by photo-crosslinking and then could be tagged with an affinity motif such as biotin by alkyne-azide click chemistry. This strategy might enable the enrichment of ligand-target peptide conjugates after digestion and purification for high resolution MS, allowing efficient mapping of biological interfaces on both sides of a ligand and a target.
Although the mass spectrometry greatly enhances the abundance of structural information obtained from the crosslinking, it often suffers from a high rate of false-positive hits and from missed true-positive hits due to complicated mass spectra to interpret, in particular when the photo-crosslinking is performed under living conditions, i.e., with high contamination backgrounds. To increase the confidence level of data analyses, a novel photo-reactive UAA bearing a transferable chemical label has been developed. The UAA introduced site-specifically to a target protein, upon interaction with a binding partner, makes a crosslink and then undergoes an oxidative cleavage leaving the label on the interface of a binding partner. This technique enables the identification of true-positive mass fragments with high confidence and the exact contact residues on both sides of interfaces simultaneously (He et al., 2017; Yang et al., 2016; Yang et al., 2017).
As demonstrated in studies reviewed above, the UAA-mediated crosslinking method genetically installs a reactive chemical handle directly into a predicted interfacial residue of interest, and induces interfacial crosslinkage of a protein complex in a controlled manner. As it require no exogenous chemical reagents at the time of crosslinking, diffusional and steric limitations that otherwise would adversely affect data fidelity can be avoided. Despite such unique advantages it provides, however, some cares need to be taken to address a potential risk of poor expression yield of UAA-incorporated proteins and/or structural perturbation of a native interface by UAA mutation. Abovementioned and related studies are summarized in the header row of ‘photo-crosslinking UAA’ (Table 1).