Structural Biology by Mass Spectrometry: Mapping Protein Interaction Surfaces of Membrane Receptor Complexes with ICAT

Abstract

Multi-component protein complexes underlie most high-order cellular functions. Elucidating the structures of these complexes and understanding how the interactions that bind them change under perturbation is a major goal of structural biology. To help in this effort, Underbakke et al provide a new twist on the proteomics technique of isotope coded affinity tagging (ICAT). In proteomics, ICAT allows relative quantification of proteins in comparable samples by reacting their exposed cysteine thiols with alkylating agents that contain either a heavy (13C) or light (12C) isotope and an affinity tag1; 2. One sample/condition is modified with the heavy tag, the other with the light, they are mixed, proteolytically cleaved into peptides, affinity purified and analyzed by mass spectrometry. Relative signals from the two mass tags report on the availability of the cysteine for modification in the two conditions. This availability reflects how much of a given cysteine residue (and its cognate protein) is there to be labeled, or in the present example, how reactive the cysteine residue is under the condition tested. Originally, the tag employed was a biotin moiety1, but this has been adapted to smaller, glucanime-based labels that can be enriched on a boronate matrix3; 4. Silverman and Harbury applied this method to the foot printing of protein accessibility and interaction surfaces4, in a manner that parallels the logic of hydrogen exchange mass spectrometery5. The current report was predicated by the author’s development of new ICAT reagents based on the glucamine backbone that are more water soluble, but have varying degrees of electrophilicity6. By measuring rates of alklyation (not just total amounts of product), the relative reactivity of cysteine residues that were specifically targeted to a protein surface could be better quantified. Here, the authors extend these studies to mapping interactions within the receptor kinase complexes that compose the bacterial chemotaxis transmembrane signaling system. Bacterial chemotaxis relys on polar clusters of transmembrane chemoreceptors coupled to the histidine kinase CheA through an adaptor protein CheW. There is great interest in the detailed architecture of the receptor arrays and how CheA activity is regulated by ligands7. Biochemical protection assays8; 9, genetic studies10; 11; 12; 13 and structural work14; 15 have probed functional interfaces in this ultrastable complex16, making it an ideal subject for testing new mapping techniques. The authors target the CheW coupling protein because it interacts with both CheA and the receptors. By substituting surface residues for cysteine and evaluating ICAT reactivity in the absence and presence of the other components they identify CheA and receptor binding regions of CheW that are largely consistent with the previous studies mentioned above. Unlike the proteomics application, careful work must be done to target the Cys residues appropriately and verify that substitution and subsequent modification does not alter activity; however, the affinity purification is a big advantage in avoiding background labeling in the complex mixture of native bacterial membranes. Importantly, the resolution of the technique is at the residue level, and has the potential to reveal how interactions between the components change subtly as they switch among activity states.