Chemical cross-linking in the structural analysis of protein assemblies

Abstract

For decades, chemical cross-linking of proteins has been an established method to study protein interaction partners. The chemical cross-linking approach has recently been revived by mass spectrometric analysis of the cross-linking reaction products. Chemical cross-linking and mass spectrometric analysis (CXMS) enables the identification of residues that are close in three-dimensional (3D) space but not necessarily close in primary sequence. Therefore, this approach provides medium resolution information to guide de novo structure prediction, protein interface mapping and protein complex model building. The robustness and compatibility of the CXMS approach with multiple biochemical methods have made it especially appealing for challenging systems with multiple biochemical compositions and conformation states. This review provides an overview of the CXMS approach, describing general procedures in sample processing, data acquisition and analysis. Selection of proper chemical cross-linking reagents, strategies for cross-linked peptide identification, and successful application of CXMS in structural characterization of proteins and protein complexes are discussed.

Keywords: Chemical cross-linking; Integrative modeling; Mass spectrometry.

Figure 1

Resolution distribution of EM maps, released up to February 2018 in the Electron Microscopy Data Bank (EMDB) (data obtained from EMStates (pdbe.org/emstats)).

Figure 2

General work-flow of chemical cross-linking and mass spectrometric analysis (CXMS) for structural characterization of proteins and protein complexes.

Figure 3

Structure models of some protein complexes: A) computational model of catalytic dimer in PDE6 holoenzyme; B) computational models of SRP•SR NG complex (colored in blue and green) superimposed with crystal structure of SRP•SR NG complex (colored in gray); C) crystal structure of HtpG, a bacterial Hsp90 homology (ADP shown as stick & ball with heteroatom color); D) computational models of MDA5 dimer in complex with viral dsRNA (cross-linked residues shown as spheres); E) computational models of human IRE1 tetramer (cross-linked residues in oligomer formation shown as spheres).

Figure 4

Reactive functionalities of some commonly used cross-linkers, including amine-reactive A) NHS-ester, B) sulfo-NHS-ester, C) imidoester (X=O) and thioimidoester (X=S), D) aldehyde; sulfhydryl-reactive E) maleimide, F) haloacetyl; carboxyl-reactive G) hydrazide, H) diazo-containing; guanidinyl-reactive I) glyoxal; photoreactive J) azide, K) diazirine, L) benzophenone; CID cleavable M) RINK group, N) Asp-Pro peptide bond; O) sulfonium, P) sulfoxide, Q) urea-linked butyrate groups.

Figure 4

Reactive functionalities of some commonly used cross-linkers, including amine-reactive A) NHS-ester, B) sulfo-NHS-ester, C) imidoester (X=O) and thioimidoester (X=S), D) aldehyde; sulfhydryl-reactive E) maleimide, F) haloacetyl; carboxyl-reactive G) hydrazide, H) diazo-containing; guanidinyl-reactive I) glyoxal; photoreactive J) azide, K) diazirine, L) benzophenone; CID cleavable M) RINK group, N) Asp-Pro peptide bond; O) sulfonium, P) sulfoxide, Q) urea-linked butyrate groups.

Figure 4

Reactive functionalities of some commonly used cross-linkers, including amine-reactive A) NHS-ester, B) sulfo-NHS-ester, C) imidoester (X=O) and thioimidoester (X=S), D) aldehyde; sulfhydryl-reactive E) maleimide, F) haloacetyl; carboxyl-reactive G) hydrazide, H) diazo-containing; guanidinyl-reactive I) glyoxal; photoreactive J) azide, K) diazirine, L) benzophenone; CID cleavable M) RINK group, N) Asp-Pro peptide bond; O) sulfonium, P) sulfoxide, Q) urea-linked butyrate groups.

Figure 5

The composition of amino acids in the UniProtKB/Swiss-Prot protein knowledgebase released on February 28^th, 2018, which includes 556,825 protein sequence entries (https://web.expasy.org/docs/relnotes/relstat.html).