Monitoring Protein Conformation Changes as an Activating Step for Protein Interactions with Cross-linking/MS Analysis. / Chen, Zhuo; Rasmussen, Morten; Tahir, Salman; Clark, C.A.C; Barlow, Paul; Rappsilber, Juri
Monitoring protein conformation changes as an activating step for protein interactions with cross-linking/MS analysis. Chen, Zhou; Rasmussen, Morten; Tahir, Salman; Clark, C.A.C; Barlow, Paul; Rappsilber, Juri. Introduction Protein interactions often require conformational changes in proteins. Chemical cross-linking of proteins coupled with mass spectrometric analysis is emerging as a versatile tool for determining low-resolution three-dimensional structures of proteins. We show in this study that this technique is also able to resolve protein conformation changes, investigating the transition of C3 to its active form C3b. C3 (187kDa) is a twelve domain protein (ANA, CUB, TED and MG1 to MG9). During the conversion of C3 to C3b, one domain, ANA (99 residues), is proteolytically removed and a number of domains change their position. This exposes protein-binding sites for downstream interactions in the complement response. Methods 250 pmol of C3 and C3b were both cross-linked with a 1000X excess of cross-linkers. BS2G, BS3, and sulfo-EGS were applied respectively. The cross-linking products were separated with 1D-PAGE gel. Monomer bands were sliced and digested with trypsin. Cross-linked peptides have been enriched with SCX-StageTips. High resolution MS/MS spectra were acquired on an LTQ-Orbitrap mass spectrometer coupled online to a nanoHPLC. Singly and doubly charged ions were rejected for fragmentation. Peak lists were generated with MaxQuant. The database searches for cross-linked peptides and manual validation were performed using in-house software. The structural information determined by validated cross-links was compared against C3 and C3b crystal structure using Pymol. Preliminary results We portray conformation changes from C3 to C3b through observing different group of cross-links in these two proteins. Moreover, there are two conflicting crystal structures for C3b, PDB|2I07 and 2HR0. The cross-linking data provides a means to determine which crystal structure is more likely to be correct. In total, we identified 46 cross-links in C3 and 43 cross-links in C3b. These cross-links were mapped into the corresponding crystal structure and are generally within the expected distance threshold, the sum of the length of two lysine side chains and cross-linker spacer-arm. We observed 29 cross-links in both samples, showing similarity between the two structures. The 31 unique cross-links identified imply the structural differences. Specifically, when the ANA domain is proteolytically removed during the conversion of C3 to C3b, seven cross-links between the ANA domain and the surrounding domains in C3 are replaced by three cross-links connected to the newly created N-terminus. The TED domain relocates as is revealed by three cross-links to the ANA domain in C3 and four cross-links to the MG1 domain in C3b, a domain that is remote in C3. In C3b case, a cross-link within the CUB domain suggests a folded structure of this domain, a matter of dispute in the competing crystal structures of C3b. Together with the positioning of the TED domain, this supports C3b structure PDB|2I07 over 2HR0. Finally, four cross-links in the C3b protein do not agree with the spatial constraints provided by either crystal structure. However, assuming the TED domain is able to rotate in solution, the cross-link data and the crystal structure can be harmonized. We suggest that cross-linking can capture aspects of protein dynamics in solution that are not observable in static crystal structures.