A Common Fold Mediates Vertebrate Defense and Bacterial Attack

Abstract

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

Perforin deficiency and susceptibility to cancer

“…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.…”

“…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.…”

Apicomplexan perforin-like proteins

“…These C-terminal domains vary in sequence and structure, but are consistently comprised almost entirely of β-pleated sheets, as in the case of the EGF-like and C2 domains of perforin, 21 the β-prism domains of the bacterial PluMACPF, 4 and the β-sandwich Ig domain of the CDCs. 22 Similarly, the C-termini of ApiPLPs are generally occupied by multiple copies of a predicted domain comprised of five β-strands, which we have termed ApiPLP C-terminal β-pleated sheet (APC-β) domains.…”

“…2,3 Exciting work over the past couple of years has provided additional insight into their molecular mechanism of action by revealing that the MACPF fold is shared with that of the cholesterol-dependent cytolysins (CDCs), an extensively studied family of bacterial pore-forming virulence factors. 4,5 Here we present an overview of the Apicomplexan PerforinLike Proteins (ApiPLPs) that have been identified in parasites for which genome sequence is available. It should be noted that apicomplexan genome sequencing efforts differ greatly in terms of completion, sequence coverage and quality of the gene predictions.…”