Abstract
Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions. Herein, we develop aromatic glyoxal cross-linkers (ArGOs) for arginine-arginine (R-R) cross-linking and the lysine-arginine (K-R) cross-linker KArGO. The R-R or K-R cross-links generated by ArGO or KArGO fit well with protein crystal structures and provide information not attainable by K-K cross-links. KArGO, in particular, is highly valuable for CXMS, with robust performance on a variety of samples including a kinase and two multi-protein complexes. In the case of the CNGP complex, KArGO cross-links covered as much of the PPI interface as R-R and K-K cross-links combined and improved the accuracy of Rosetta docking substantially.
Highlights
Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics
In order to gauge the potential advantages of arginine-selective cross-linkers for structural biology, we analysed the number of theoretical cross-linkable Arg–Arg and Lys–Arg pairs across 1808 PDB complexes, using a method described before[30]
We have developed a series of homo-bifunctional aromatic glyoxals (ArGOs) that cross-link proteins selectively at arginine residues; and a hetero-bifunctional cross-linker (KArGO) that targets both lysine and arginine
Summary
We performed CXMS analyses of a UtpA subcomplex containing subunits Utp[4, 5, 8, 9], and 15, and identified a total of 22 and 14 high-confidence (best E-value < 0.001, ≥6 spectra) inter-protein cross-links with BS3 and KArGO, respectively (Fig. 4a and Supplementary Table 4). Mapping these inter-protein cross-links onto the cryo-EM structure of UtpA61 we find that the K–R cross-links greatly complemented the K–K cross-links and provided rich information about the interface between subunits. These data strongly suggest that in the UtpA sub-complex analysed in this study, the Utp[4] subunit takes a position that is closer to Utp[15] and Utp[5] than it does in the UtpA complex that had been analysed by cryo-EM
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