Abstract
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.
Highlights
The human immune system plays a crucial role in detecting and clearing bacterial infections
The membrane attack complex (MAC) components were more enriched in the wt strain than in the M1 mutant (Figure 1C), consistent with MAC binding to S. pyogenes being complement C3-independent
We previously showed the power of TX-mass spectrometry (MS) method, which is a combinatorial approach based on different MS acquisition techniques and macromolecular modeling to study host– pathogen interactions (Hauri et al, 2019)
Summary
The human immune system plays a crucial role in detecting and clearing bacterial infections. Complement activation can occur through three distinct pathways depending on the molecular trigger, all proceeding through a proteolytic cascade that leads to the formation of the multicomponent membrane attack complex (MAC) capable of disrupting target cell membranes. While Gram-positive bacteria are thought to be able to resist the MAC-induced membrane disruption due to their thick cell wall, several such bacteria have been shown to secrete small proteins that target this multicomponent. Berends et al (2013) found that complement activation leads to specific C3independent deposition of MAC on the Gram-positive bacterial surface. The mammalian peptidoglycan recognition proteins (PGRPs) bind to the bacterial cell wall of Gram-positive Bacillus subtilis and kill the bacteria through specific interaction with the CssR–CssS two-component system at the site of daughter cell separation (Kashyap et al, 2011). While we previously reported interactions between MAC components and bacterial CovR, part of CovRS two-component system in S. pyogenes (Happonen et al, 2019), the importance of studying the MAC formation on the bacterial surface is evident
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