Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes

The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.Proteins exert the majority of their functions in the form of protein complexes to control cellular signaling, protein synthesis, folding and degradation, and many more essential processes. Therefore, elucidating the composition and structure of such complexes has been a longstanding goal of biological research.MS-based proteomics has emerged as one of the main techniques to identify and quantify proteins and their modifications in biological samples such as isolated complexes, proteome fractions, or whole proteomes. Various MS methods now provide structural information on protein assemblies (1–3). Among them, chemical cross-linking and identification of cross-linked peptides by MS (XL-MS) has been increasingly applied to determine the subunit arrangements of biologically relevant complexes (4–6). Such XL-MS experiments indicate the locations of cross-linking sites and thus the spatial proximity of reactive groups that are connected by a covalent bond. This information is then used to determine the positioning of subunits or locate interacting regions, alone or in combination with other techniques such as NMR spectroscopy, electron microscopy, and X-ray crystallography.In the last few years, optimized protocols and new computational tools for the reliable analysis of XL-MS datasets resulted in significant advances of the XL-MS technology (4–6). These advances have contributed to the emergence of a robust, integrated XL-MS method that has been successfully applied for structure determination of a number of large protein complexes (7–11) and the detection of direct, physical interactions in whole cells (12–14). To date, the cross-linking chemistries applied in these studies have targeted primary amines. Predominantly, N-hydroxysuccinimide esters were used as reactive groups, although other chemistries, for example, based on amidates, have also been described (8, 15, 16). Cysteine-specific cross-linking is also well established but usually does not yield sufficient structural information due to the low prevalence of cysteines in proteins and their involvement in the formation of disulfide bonds. Zero-length cross-linking by carbodiimide coupling (17–19) and photochemical cross-linking (20) are other strategies that have been described but have not yet found widespread application in the field.The development of cross-linking chemistries that cross-link functional groups different from amino groups but maintain the efficiency achieved by amine-specific cross-linking are expected to be highly beneficial to increase the depth of structural information obtained from cross-linking experiments. Specifically, such a technique would generate distance restraints from protein regions that are refractive to amine-specific cross-linking under the conditions used and reduce the coverage bias in basic sites. Also, typically only a fraction of theoretically possible cross-links are experimentally observed. This effect is presumably caused by variations in the reactivity of individual lysine residues and/or the unsuitable properties of the resulting cross-linked peptides for MS analysis. An increase in the number of cross-links per substrate by the use of an orthogonal chemistry would therefore be beneficial for de novo identification of hetero-oligomer subunit architectures, as well as restraints in hybrid methods incorporating low-resolution structural information.Residues with carboxyl-terminating side-chains (aspartic and glutamic acids) are attractive targets because of their high prevalence in most proteins. In the most recent release of the SwissProt database (21), version 2014_04, 5.5% and 6.7% of all residues are Asp and Glu, respectively, compared with 5.8% for Lys. However, the low intrinsic chemical reactivity of carboxylic acids poses practical challenges for cross-linking reactions, requiring the use of a coupling reagent. Novak and Kruppa used different dihydrazides as cross-linking reagents in combination with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for activation (22). However, to obtain sufficiently high reaction yields, cross-linking was carried out at relatively low pH (5.5 in ref. 22), which is incompatible with pH-sensitive assemblies. Furthermore, the method was only applied to a single protein, ubiquitin, resulting in the identification of two cross-links.Here we introduce a cross-linking chemistry that connects proximal carboxyl groups [acidic cross-linking (AXL)], whereby side-chains of Asp and Glu residues are cross-linked with dihydrazides using the coupling reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (23) (Fig. 1A). In contrast to EDC, DMTMM is able to couple carboxylic acids with hydrazide-based cross-linkers at neutral pH (7–7.5), ensuring good reaction yields and biocompatibility. Experiments with model proteins yielded numbers of cross-linked peptides that were in the same range to those generated by the well-established Lys-specific cross-linking using the reagent disuccinimidyl suberate (DSS). On top of that, a second set of cross-linking restraints is observed in the form of zero-length cross-links between Lys and Asp or Glu residues, respectively.Open in a separate windowFig. 1.(A) Cross-linking reactions involving coupling of carboxyl groups using dihydrazides (acidic cross-linking, AXL; Top) and zero-length cross-linking (ZLXL) with DMTMM as coupling reagent. R¹ and R² denote acidic residues (Asp, Glu) in a single or two different proteins, R³ denotes a primary amine (usually Lys). (B) Structure of the two dihydrazide reagents used in this study. Asterisks denote positions where hydrogen atoms are exchanged for deuterium atoms in the heavy form of the reagent. The spacer length of the reagents (calculated between the terminal nitrogen atoms) is also given.To show the practical relevance of the method, we applied it to multisubunit complexes in the megadalton range that have been recently probed with lysine-specific cross-linking and for which structural information is available. The results indicate that for both the chaperonin TRiC/CCT from Bos taurus and the Schizosaccharomyces pombe 26S proteasome, cross-links were identified that are in agreement with the available structures of these complexes. Acidic and zero-length cross-links provided orthogonal sets of structural restraints that are complementary to a cross-linking chemistry targeting lysines. We therefore expect that chemical cross-linking of acidic residues will become an important method for a more comprehensive structural analysis of protein complexes by XL-MS.