Rapid Mapping of Protein Interactions Using Tag‐Transfer Photocrosslinkers

Abstract

Analysing protein complexes by chemical crosslinking‐mass spectrometry (XL‐MS) is limited by the side‐chain reactivities and sizes of available crosslinkers, their slow reaction rates, and difficulties in crosslink enrichment, especially for rare, transient or dynamic complexes. Here we describe two new XL reagents that incorporate a methanethiosulfonate (MTS) group to label a reactive cysteine introduced into the bait protein, and a residue‐unbiased diazirine‐based photoactivatable XL group to trap its interacting partner(s). Reductive removal of the bait transfers a thiol‐containing fragment of the crosslinking reagent onto the target that can be alkylated and located by MS sequencing and exploited for enrichment, enabling the detection of low abundance crosslinks. Using these reagents and a bespoke UV LED irradiation platform, we show that maximum crosslinking yield is achieved within 10 seconds. The utility of this “tag and transfer” approach is demonstrated using a well‐defined peptide/protein regulatory interaction (BID80‐102/MCL‐1), and the dynamic interaction interface of a chaperone/substrate complex (Skp/OmpA).

The Application of Fluorine‐Containing Reagents in Structural Proteomics

“…In contrast to af ootprinter that contains one reactive group,cross-linking reagents comprise two or more functional groups that spatially tether amino acid residues through the formation of covalent bonds.T he goal of cross-linking is to determine the topology of protein complexes and identify adjoining regions.T he length of the cross-linker is as patial restraint for the distance between two reactive sites,and some cross-linking studies use avariety of linkers.The development of cross-linking reagents follows two main trends:1)improving chemical reactivities to capture more amino acid sites,and 2) increasing the identification confidence and sensitivity to detect low-abundance cross-linked products.I nt his regard, strategies such as cleavable cross-linkers,a ffinity tags for cross-link enrichment, and heterobifunctional cross-linkers with photoactivable headgroups are valuable.T he field of cross-linking was reviewed by Sinz. [54] Fluorine-containing cross-linkers have been described by Radford, Wilson, and co-workers, [55] who developed an ovel "tag and transfer" approach that incorporates amethanethiosulfonate (MTS) group and ap hotoactivable diazirine group (one of them aT PD) into ah eterobifunctional photo-crosslinker.T he MTS group first reacts with as ingle Cys residue introduced into the "bait" protein, thereby fixing the crosslinker with the "bait" protein. Subsequent incubation allows the target protein to form ac omplex, which is followed by photochemical cross-linking between these two proteins.T he "bait" protein is removed by reduction of ad isulfide bond with SDS-PAGE( Figure 12).…”

Fluorierte Reagenzien in der Strukturproteomik

“…[46,50] [54] Fluorierte Crosslinker wurden von Radford und Mitarbeitern beschrieben. [55] Sie entwickelten einen neuen Ansatz namens "tag and transfer", der eine Methanthiosulfonat (MTS)-Gruppe und eine photoaktivierbare Diaziringruppe (eine davon ist ein TPD) in einen heterodifunktionalen Crosslinker einbaut. Die MTS-Gruppe reagiert zuerst mit einem in einem "Kçder"-Protein ( Ein Ziel in der Strukturproteomik ist die Bestimmung der endogenen Proteinstruktur in lebenden Zellen.…”

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

“…To probe the organisation of the SurA-OmpX complex in more detail we next exploited the ability of the photoactivatable cross-linker MTS-diazirine ( Supplementary Fig. 9a) to react rapidly (within ns 45 ), and non-specifically with any residue within ~15 Å of the diazirine moiety (Cα-Cα Euclidean distance) 46 . This "tag transfer" method was developed specifically to enable detection of weak and transient protein-protein interactions 46 .…”

“…9a) to react rapidly (within ns 45 ), and non-specifically with any residue within ~15 Å of the diazirine moiety (Cα-Cα Euclidean distance) 46 . This "tag transfer" method was developed specifically to enable detection of weak and transient protein-protein interactions 46 . We created four MTS-diazirine-labelled single-Cys variants of OmpX (M41C, I102C, K122C, V167C), formed complexes of each with SurA, and following rapid UV irradiation (for only 30 sec) 46 , identified the crosslinked products by LC-MS/MS ( Fig.…”

“…9a-c and Supplementary Table 8) 46 . Despite the location of the labelled Cys residues in distant regions of the OmpX sequence and the lack of specificity of the diazirine crosslinker 46 , all four Cys-OmpX variants crosslinked to the N-terminal domain of SurA (11 crosslinked sites were identified, Supplementary Table 8) and P1 (two crosslinked sites), indicating that these regions form the heart of the binding epitope ( Fig. 5b,c, Supplementary Fig.…”