Integral β-barrel proteins are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. The assembly of these proteins requires a proteinaceous apparatus of which Omp85 is an evolutionary conserved central component. To study its molecular mechanism, we have produced Omp85 from Escherichia coli in inclusion bodies and refolded it in vitro. The interaction of Omp85 with its substrate proteins was studied in lipid-bilayer experiments, where it formed channels. The properties of these channels were affected upon addition of unfolded outer-membrane proteins (OMPs) or synthetic peptides corresponding to their C-terminal signature sequences. The interaction exhibited species specificity, explaining the inefficient assembly of OMPs from Neisseria in E. coli. Accordingly, the in vivo assembly of the neisserial porin PorA into the E. coli outer membrane was accomplished after adapting its signature sequence. These results demonstrate that the Omp85 assembly machinery recognizes OMPs by virtue of their C-terminal signature sequence.

Regulators of complement activation (RCA) inhibit complement‐induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i–iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b‐binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease‐related mutations and immune evasion. Synopsis Complement activity is controlled by regulators binding to C3b or C4b. Analysis of five C3b regulator structures reveals that both decay‐acceleration and cofactor activity depend on a common C3b‐regulator binding mode. Crystal structures reveal C3b binding of four human and viral complement regulators Common mode of C3b binding underlies decay‐acceleration and cofactor activity Extensive variations in interaction details explain diverse characteristics of regulators C3b‐regulator structures provide basis for understanding disease‐related mutations

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. ItsEscherichia colihomolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the β-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components inNeisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As inE. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by itsE. colihomolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. AbamCmutant showed only marginal OMP assembly defects, but the impossibility of creating abamC bamEdouble mutant further indicated the function of BamC in OMP assembly. Anmlpmutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex inN. meningitidisconsists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.

Atypical HUS (aHUS) is thought to be caused by predisposing mutations in genes encoding complement (regulating) proteins, such as Factor H (CFH), Factor I (IF), membrane co-factor protein (MCP) and Factor B (FB), or by auto-antibodies against CFH (alphaFH) in combination with a homozygous polymorphic deletion of the genes encoding Complement Factor H-related 1 and 3 (DeltaCFHR1/3). The clinical impact of this knowledge is high, as it might be a prognostic factor for the outcome of renal transplantations and kidney donations. Mutational screening, by means of PCR and DNA sequencing, is performed in the above-mentioned genes in a group of 72 aHUS patients. Also, the presence of alphaFH and DeltaCFHR1/3 was tested in patients and controls. In 23 patients, a genetic aberration in at least one gene or the presence of alphaFH was found. A heterozygous mutation was observed in CFH in nine patients, in IF in seven patients and in MCP in three patients. No mutations were observed in FB. Seven patients presented alphaFH, of whom five also carried DeltaCFHR1/3. Three patients carried a combined mutation (two patients: IF and MCP; one patient: IF, alphaFH and DeltaCFHR1/3). A significant difference between patients and controls was detected for the presence of all three associated polymorphisms in CFH. Genetic abnormalities or the presence of alphaFH were detected in 31.9% of the aHUS patients. Furthermore, bigenic mutations were present, indicating that routine DNA mutation analysis of all complement factors associated with aHUS is important.

Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in the Western world. While AMD is a multifactorial disease, the complement system was identified as one of the main pathways contributing to disease risk. The strong link between the complement system and AMD was demonstrated by genetic associations, and by elevated complement activation in local eye tissue and in the systemic circulation of AMD patients. Several complement inhibitors have been and are being explored in clinical trials, but thus far with limited success, leaving the majority of AMD patients without treatment options to date. This indicates that there is still a gap of knowledge regarding the functional implications of the complement system in AMD pathogenesis and how to bring these towards clinical translation. Many different experimental set-ups and disease models have been used to study complement activation in vivo and in vitro, and recently emerging patient-derived induced pluripotent stem cells and genome-editing techniques open new opportunities to study AMD disease mechanisms and test new therapeutic strategies in the future. In this review we provide an extensive overview of methods employed to understand the molecular processes of complement activation in AMD pathogenesis. We discuss the findings, advantages and challenges of each approach and conclude with an outlook on how recent, exciting developments can fill in current knowledge gaps and can aid in the development of effective complement-targeting therapeutic strategies in AMD.