Cross‐linking mass spectrometry has developed into an important method to study protein structures and interactions. The in‐solution cross‐linking workflows involve time and sample consuming steps and do not provide sensible solutions for differentiating cross‐links obtained from co‐occurring protein oligomers, complexes, or conformers. Here we developed a cross‐linking workflow combining blue native PAGE with in‐gel cross‐linking mass spectrometry (IGX‐MS). This workflow circumvents steps, such as buffer exchange and cross‐linker concentration optimization. Additionally, IGX‐MS enables the parallel analysis of co‐occurring protein complexes using only small amounts of sample. Another benefit of IGX‐MS, demonstrated by experiments on GroEL and purified bovine heart mitochondria, is the substantial reduction of undesired over‐length cross‐links compared to in‐solution cross‐linking. We next used IGX‐MS to investigate the complement components C5, C6, and their hetero‐dimeric C5b6 complex. The obtained cross‐links were used to generate a refined structural model of the complement component C6, resembling C6 in its inactivated state. This finding shows that IGX‐MS can provide new insights into the initial stages of the terminal complement pathway.

Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane β-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.

A major cause of visual impairment, corneal dystrophies result from accumulation of protein deposits in the cornea. One of the proteins involved is transforming growth factor β-induced protein (TGFBIp), an extracellular matrix component that interacts with integrins but also produces corneal deposits when mutated. Human TGFBIp is a multi-domain 683-residue protein, which contains one CROPT domain and four FAS1 domains. Its structure spans ∼120 Å and reveals that vicinal domains FAS1-1/FAS1-2 and FAS1-3/FAS1-4 tightly interact in an equivalent manner. The FAS1 domains are sandwiches of two orthogonal four-stranded β sheets decorated with two three-helix insertions. The N-terminal FAS1 dimer forms a compact moiety with the structurally novel CROPT domain, which is a five-stranded all-β cysteine-knot solely found in TGFBIp and periostin. The overall TGFBIp architecture discloses regions for integrin binding and that most dystrophic mutations cluster at both molecule ends, within domains FAS1-1 and FAS1-4.

Transforming growth factor-β-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-β-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.