TY - JOUR AU - Khakzad, Hamed AU - Happonen, Lotta AU - Tran Van Nhieu, Guy AU - Malmström, Johan AU - Malmström, Lars PY - 2021 M3 - Original Research TI - In vivo Cross-Linking MS of the Complement System MAC Assembled on Live Gram-Positive Bacteria JO - Frontiers in Genetics UR - https://www.frontiersin.org/articles/10.3389/fgene.2020.612475 VL - 11 SN - 1664-8021 N2 - Protein–protein interactions are central in many biological processes, but they are challenging to characterize, especially in complex samples. Protein cross-linking combined with mass spectrometry (MS) and computational modeling is gaining increased recognition as a viable tool in protein interaction studies. Here, we provide insights into the structure of the multicomponent human complement system membrane attack complex (MAC) using in vivo cross-linking MS combined with computational macromolecular modeling. We developed an affinity procedure followed by chemical cross-linking on human blood plasma using live Streptococcus pyogenes to enrich for native MAC associated with the bacterial surface. In this highly complex sample, we identified over 100 cross-linked lysine–lysine pairs between different MAC components that enabled us to present a quaternary model of the assembled MAC in its native environment. Demonstrating the validity of our approach, this MAC model is supported by existing X-ray crystallographic and electron cryo-microscopic models. This approach allows the study of protein–protein interactions in native environment mimicking their natural milieu. Its high potential in assisting and refining data interpretation in electron cryo-tomographic experiments will be discussed. ER -