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

Abstract

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.

Fig. 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.

Fig. 2.

Experimental workflow for the chemical cross-linking of acidic side-chains and the identification of cross-linked peptides by MS.

Fig. 3.

Histogram depicting the distribution of C_α-C_α distances as obtained by validation of structures of the model proteins. Data are shown for the acidic cross-linking reagents ADH (green) and PDH (magenta), zero-length cross-links (ZLXL, from ADH experiment, in orange), and previous data from a lysine-lysine cross-linking study using DSS (blue; taken from ref. 28).

Fig. 4.

Cross-linking of acidic residues and zero-length cross-linking applied to the TRiC/CCT and 26S proteasome complexes. (A) Connectivity map for TRiC/CCT depicting the intersubunit contacts observed. (B) Overview of intersubunit cross-links observed in different regions of the 26S proteasome. Structures of the complexes are used for illustratory purposes only.