Crystal Structure of Human Complement Protein C8gamma with Laurate (original) (raw)
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Biochimica Et Biophysica Acta-proteins and Proteomics, 2007
Human C8 is one of five components of the cytolytic membrane attack complex of complement. It contains three subunits (C8α, C8β, C8γ) arranged as a disulfide-linked C8α-γ heterodimer that is noncovalently associated with C8β. C8γ has the distinction of being the only lipocalin in the complement system. Lipocalins have a core β-barrel structure forming a calyx with a binding site for a small hydrophobic ligand. A natural ligand for C8γ has not been identified; however previous structural studies indicate C8γ has a typical lipocalin fold that is suggestive of a ligandbinding capability. A distinctive feature of C8γ is the division of its putative ligand binding pocket into a hydrophilic upper portion and a large hydrophobic lower cavity. Access to the latter is restricted by the close proximity of two tyrosine side chains (Y83 and Y131). In the present study, binding experiments were performed using lauric acid as a pseudoligand to investigate the potential accessibility of the lower cavity. The crystal structure of a C8γ·laurate complex revealed that Y83 and Y131 can move to allow penetration of the hydrocarbon chain of laurate into the lower cavity. Introducing a Y83W mutation blocked access but had no effect on the ability of C8γ to enhance C8 cytolytic activity. Together, these results indicate that the lower cavity in C8γ could accommodate a ligand if such a ligand has a narrow hydrophobic moiety at one end. Entry of that moiety into the lower cavity would require movement of Y83 and Y131, which act as a gate at the cavity entrance.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006
Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the membrane attack complex (MAC). C8 is composed of a disulfide-linked C8α-γ heterodimer and a noncovalently associated C8β chain. C8α and C8β are homologous to C6, C7 and C9, whereas C8γ is the only lipocalin in the complement system. Lipocalins have a core β-barrel structure forming a calyx with a binding site for a small molecule. In C8γ, the calyx opening is surrounded by four loops that connect β-strands. Loop 1 is the largest and contains Cys40 that links to Cys164 in C8α. To determine if these loops mediate binding of C8α prior to interchain disulfide bond formation in C8α-γ, the loops were substituted separately and in combination for the corresponding loops in siderocalin (NGAL, Lcn2), a lipocalin that is structurally similar to C8γ. The siderocalin-C8γ chimeric constructs were expressed in E. coli, purified, and assayed for their ability to bind C8α. Results indicate at least three of the four loops surrounding the entrance to the C8γ calyx are involved in binding C8α. Binding near the calyx entrance suggests C8α may restrict and possibly regulate access to the C8γ ligand binding site.
Journal of Molecular Biology, 2008
Human 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 ). 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.
Journal of Molecular Biology, 2010
Human 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 ). 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.
Journal of Biological Chemistry, 2011
C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like "membrane attack complex" (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12-18 molecules of C9. C8 is composed of three genetically distinct subunits, C8␣, C8, and C8␥. The C6, C7, C8␣, C8, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N-and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8␣ and C8 are located on the periphery of C8 and not likely to interact with the target membrane. The C8␥ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8␣ and C8 are related by a rotation of ϳ22°with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane -hairpins to form a circular pore.
Structure of the poly-C9 component of the complement membrane attack complex
Nature communications, 2016
The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane inser...
Biochemical and Biophysical Research Communications, 1987
Anti-C8a-y specific antibodies were used to isolate cDNA clones from a human liver expression library. Antibodies affinity-purified on the expressed hybrid protein of one clone bound exclusively to the y-chain of reduced C8a-y. This clone, as well as a second full length cDNA clone obtained by hybridization screening, were sequenced and the complete primary structure for C8r was established. Cyanogen bromide cleavage of CSa-y released a 12 kDa carboxy-terminal C8y fragment under both reducing and nonreducing conditions which was identified by fragmentspecific, affinity-purified antibodies. Our data clearly show that C8r has one internal disulfide bridge between cys-76 and cys-168 within the carboxy-terminal 12 kDa fragment, whereas the remaining cysteine residue 40 forms the disulfide bridge with C8a. The overall sequence homology to plasma protein HC (23% amino acid identities) and the conservation of one internal cysteine bond and one free, surface-located cysteine residue suggests a highly conserved threedimensional structure of C8y and protein HC and also a possible functional relationship between these proteins. 0 1987 Academic Press, Inc.
X-ray crystal structure of the C4d fragment of human complement component C4
Journal of molecular biology, 2002
C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3 Å resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the , 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase.
Molecular Immunology, 2002
C1 is the multimolecular protease that triggers activation of the classical pathway of complement, a major element of antimicrobial host defense also involved in immune tolerance and various pathologies. This 790 000 Da complex is formed from the association of a recognition protein, C1q, and a catalytic subunit, the Ca 2+ -dependent tetramer C1s-C1r-C1r-C1s comprising two copies of each of the modular proteases C1r and C1s. Early studies mainly based on biochemical analysis and electron microscopy of C1 and its isolated components have allowed for characterization of their domain structure and led to a low-resolution model of the C1 complex in which the elongated C1s-C1r-C1r-C1s tetramer folds into a more compact, "8-shaped" conformation upon interaction with C1q. A major strategy used over the past years has been to dissect the C1 proteins into modular segments to characterize their function and solve their structure by either X-ray crystallography or nuclear magnetic resonance spectroscopy (NMR). The purpose of this review is to focus on this information, with particular emphasis on the architecture of the C1 complex and the mechanisms underlying its activation and proteolytic activity.
The first transmembrane region of complement component-9 acts as a brake on its self-assembly
Nature communications, 2018
Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.