Crystal Structure of the MACPF Domain of Human Complement Protein C8α in Complex with the C8γ Subunit

Abstract

SummaryHuman C8 is one of five complement components (C5b, C6, C7, C8 and C9) that assemble on bacterial membranes to form a pore-like structure referred to as the "membrane attack complex" (MAC). C8 contains three genetically distinct subunits (C8α, C8β, Cγ.) arranged as a disulfide-linked C8α-γ dimer that is noncovalently associated with C8β. C6, C7 C8α, C8β and C9 are homologous. All contain N-and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. The C8γ subunit is unrelated and belongs to the lipocalin family of proteins that display a β-barrel fold and generally bind small, hydrophobic ligands. Several hundred proteins with MACPF domains have been identified based on sequence similarity; however, the structure and function of most are unknown. Crystal structures of the secreted bacterial protein Plu-MACPF and the human C8α MACPF domain were recently reported and both display a fold similar to the bacterial pore-forming cholesterol-dependent cytolysins (CDC). In the present study, we determined the crystal structure of the human C8α MACPF domain disulfide-linked to C8γ (αMACPF-γ) at 2.15 Å resolution. The αMACPF portion has the predicted CDC-like fold and shows two regions of interaction with C8γ. One is in a previously characterized 19-residue insertion (indel) in C8α and fills the entrance to the putative C8γ ligand binding site. The second is a hydrophobic pocket that makes contact with residues on the side of the C8γ β-barrel. The latter interaction induces conformational changes in αMACPF that are likely important for C8 function. Also observed is structural conservation of the MACPF signature motif Y/W-G-T/S-H-F/Y-X 6 -G-G in αMACPF and Plu-MACPF, and conservation of several key glycine residues known to be important for refolding and pore formation by CDCs. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.Accession code -Protein Data Bank: αMACPF-γ atomic coordinates together with structure factors have been deposited with accession code 2RD7.

Temperature sensitivity of human perforin mutants unmasks subtotal loss of cytotoxicity, delayed FHL, and a predisposition to cancer

“…S1), it seemed puzzling that many of the patients (Patients 1, 2, 12-15, 17, and 18) had escaped FHL in infancy and in some cases, survived well for many years. To determine whether the mutations had any common feature, we first modeled their locations on the crystal structures of several PRF-like proteins that we and others solved recently (21)(22)(23). We were surprised to discover that the 4 mutations hA91V, hF193L, hP201T, and hR356W, present at least once in 18 of the 23 patients, were clustered within adjacent helices G and H of the MACPF domain.…”

“…We noted recently that pore formation by PRF-like toxins relies heavily on major conformational changes because they function in an analogous fashion to the bacterial cholesterol-dependent cytolysins and undergo substantial rearrangement to form transmembrane pores (21)(22)(23). Molecules that require marked conformational change for their function are often susceptible to mutations that impose an inappropriate conformation (25).…”

Perforin deficiency and susceptibility to cancer

“…However, in the year 2007-2008, three independent studies arrived at the same remarkable conclusion: despite sharing minimal amino acid sequence similarity, mammalian and other MACPF proteins are structurally related to bacterial cholesterol-dependent cytolysins (CDCs) and operate by an analogous mechanism. [31][32][33] These studies opened new horizons in studying MACPF proteins, as their membership with a large family of well-characterized proteins has finally been identified. Despite these advances, major milestones in MACPF research are yet to be reached, namely, the crystal structure of a pore-forming MACPF protein, the mechanism of pore formation and the structure of a pore.…”

“…34 Importantly, this discovery indirectly assigned the membrane-spanning role to two alternative helical domains TMH1 and TMH2 that unfold into two membrane-spanning b-hairpins. [31][32][33] This provided (2) It has been hypothesized that binding and activation of the high-affinity Ca 2 þ -dependent phospholipid C2 domain of PRF is prevented in the ER (which has high Ca 2 þ and neutral pH) by the extreme C-terminus (last 12-20 a.a.). (3) The extreme C-terminus is proteolytically cleaved in the acidic SGs, which is thought to liberate the Ca 2 þ -binding moiety and produce a form of PRF that remains nonfunctional until the pH becomes neutral in the context of the immune synapse.…”

“…Our initial analysis suggested that all but 3 of 17 cancer-and late FHL-associated PRF mutants had no cytotoxic function when analyzed in vitro. However we also discovered, through mapping these mutations onto the predicted three dimension PRF structure (based on three crystallized MACPF proteins [31][32][33] ) that many of the mutants localized to a single subdomain at the top of the PRF monomer, the part furthest removed from the membrane-binding domains. 10 Intriguingly, in light of previous studies on A91V, 80 this common variant was also shown to map to the same subdomain and result in protein misfolding.…”