Membrane pore formation by human complement: Functional importance of the transmembrane β-hairpin (TMH) segments of C8α and C9

Abstract

Human C8 and C9 have a key role in forming the pore-like “membrane attack complex” (MAC) of complement on bacterial cells. A possible mechanism for membrane insertion of these proteins was suggested when studies revealed a structural similarity between the MACPF domains of the C8α and C8β subunits and the pore-forming bacterial cholesterol-dependent cytolysins (CDCs). This similarity includes a pair of α-helical bundles that in the CDCs refold during pore formation to produce two transmembrane β-hairpins (TMH1 and TMH2). C9 is the major pore-forming component of the MAC and is also likely to contain two TMH segments because of its homology to C8α and C8β. To determine their potential for membrane insertion, the TMH sequences in C8α and those predicted to be in C9 were substituted for the TMH sequences in perfringolysin O (PFO), a well-characterized CDC. Only chimeric proteins containing TMH2 from C8α (PFO/αT2) or C9 (PFO/C9T2) could be expressed in soluble, active form. The PFO/αT2 and PFO/C9T2 chimeras retained significant hemolytic activity, formed pore-like structures on membranes, and could combine with PFO to form hemolytically active mixed complexes that were functionally similar to PFO alone. These results provide experimental evidence in support of the hypothesis that TMH segments in C8α and those predicted to be in C9 have a direct role in MAC membrane penetration and pore formation.

Comprehensive Proteoform Characterization of Plasma Complement Component C8αβγ by Hybrid Mass Spectrometry Approaches

“…Interestingly, the C8β N243 glycosylation is in the C8β MACPF domain. These MACPF domains undergo a large conformational change when the soluble C8 is incorporated into the MAC [ 42 ]. Especially, the transmembrane β-hairpin (TMH) segments play a direct role in MAC membrane penetration.…”

The Making of a Macromolecular Machine: Assembly of the Membrane Attack Complex

“…Recent experiments involving chimeric complement proteins provide further experimental evidence that the predicted TMH segments of C8 and C9 are directly involved in membrane penetration and pore formation. 44 Cryo-EM reconstructions of CDC prepore and pore complexes 45 together with fluorescence-based biophysical data 46 have provided key insights into how MACPF domains are arranged in membranes (Figure 4C). For the majority of CDCs, association with cholesterol through domain 4 (lacking in complement components) is allosterically coupled to large conformational changes in the CDC/MACPF domain during pore formation.…”

“…The segments in C9 are longer still and possess a hydrophilic loop at the tip, which may interact with intracellular proteins or anchor the pore to the cytoplasm. Recent experiments involving chimeric complement proteins provide further experimental evidence that the predicted TMH segments of C8 and C9 are directly involved in membrane penetration and pore formation …”

Proteomics Analysis of Preeclampsia, a Systematic Review of Maternal and Fetal Compartments

“…Complement activation by the complement component 3 (C3) and complement component 5a (C5a) fragments are associated with pregnancy loss showing complement activation can adversely affect pregnancy. C8 and C9 are activated in the final steps to form pores on target cells and induce cell death by contributing to the membrane attack complex (MAC) [24]. Complement component 8 (C8) and complement component (C9) were observed as ambiguously regulated in two studies, one serum source reported C8 and C9 as down regulated at delivery while the other reported up-regulated but the gestational age of the samples was not reported (Table2 and Supplementary Table 1) also ambiguous was Complement factor H-1 related protein, which was previously identified as a potential biomarker for preeclampsia [4].…”