Mapping the Active Site of CD59 (original) (raw)
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Insights into the Human CD59 Complement Binding Interface Toward Engineering New Therapeutics
Journal of Biological Chemistry, 2005
CD59 is a 77-amino acid membrane glycoprotein that plays an important role in regulating the terminal pathway of complement by inhibiting formation of the cytolytic membrane attack complex (MAC or C5b-9). The MAC is formed by the self assembly of C5b, C6, C7, C8, and multiple C9 molecules, with CD59 functioning by binding C5b-8 and C5b-9 in the assembling complex. We performed a scanning alanine mutagenesis screen of residues 16-57, a region previously identified to contain the C8/C9 binding interface. We have also created an improved NMR model from previously published data for structural understanding of CD59. Based on the scanning mutagenesis data, refined models, and additional site-specific mutations, we identified a binding interface that is much broader than previously thought. In addition to identifying substitutions that decreased CD59 activity, a surprising number of substitutions significantly enhanced CD59 activity. Because CD59 has significant therapeutic potential for the treatment of various inflammatory conditions, we investigated further the ability to enhance CD59 activity by additional mutagenesis studies. Based on the enhanced activity of membrane-bound mutant CD59 molecules, clinically relevant soluble mutant CD59-based proteins were prepared and shown to have up to a 3-fold increase in complement inhibitory activity. Complement is an important component of host defense and is an effector mechanism for both innate and adaptive immune responses. Complement also plays important roles in enhancing the induction of both humoral and cellular immunity, regulating tolerance to self-antigens, and in the clearance of immune complexes and apoptotic cells. These effects of complement are mediated either directly or indirectly by bioactive cleaved protein fragments or by a terminal cytolytic protein assembly, termed the membrane attack complex (MAC 3 or C5b-9). Generation of the MAC during the complement cascade is initiated by cleavage of C5, which yields C5b and results in the sequential binding of C6, C7, C8, and multiple C9 molecules. Necessarily, complement effector mechanisms are under tight control to prevent damage to host cells, and MAC formation is under the control of CD59, a widely distributed 18-21-kDa (77 amino acids) glycoprotein attached to the plasma mem
Membrane defence against complement lysis: The structure and biological properties of CD59
Immunologic Research, 1993
The complement system is an important branch of the innate immune response, constituting a first line of defence against invading microorganisms which activate complement via both antibody-dependent and -independent mechanisms. Activation of complement leads to (a) a direct attack upon the activating cell surface by assembly of the pore-forming membrane attack complex (MAC), and (b) the generation of inflammatory mediators which target and recruit other branches of the immune system. However, uncontrolled coinplement activation can lead to widespread tissue damage in the host, since certain of the activation products, notably the fragment C3b and the C5b-7 complex, can bind nonspecifically to any nearby cell membranes. Therefore it is important that complement activation is tightly regulated. Our own cells express a number of membrane-bound control proteins which limit complement activation at the cell surface and prevent accidental complement-mediated damage. These include decay-accelerating factor, complement receptor 1 and membrane cofactor protein, all of which are active at the level of C3/C5 convertase formation. Until recently, cell surface control of MAC assembly had been attributed to a single 65-kD membrane protein called homologous restriction factor (alternatively named CS-binding protein and MAC-inhibiting protein). However a second MAC-inhibiting protein has since been discovered and it is now clear that this protein plays a major role in the control of membrane attack. This review charts the rapid progress made in elucidating the protein and gene structure, and the mechanism of action of this most recently discovered complement inhibitor, CD59. Dr. Alexandra Davies 9 1993 MRC Centre S. Karger AG, Basel Hills Road 0257-277X/93/ Cambridge CB2 2QH (UK) 0123-025852.75/0
Journal of Biological Chemistry, 2011
CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.
Cell reports, 2013
Pore-forming proteins containing the structurally conserved membrane attack complex/perforin fold play an important role in immunity and host-pathogen interactions. Intermedilysin (ILY) is an archetypal member of a cholesterol-dependent cytolysin subclass that hijacks the complement receptor CD59 to make cytotoxic pores in human cells. ILY directly competes for the membrane attack complex binding site on CD59, rendering cells susceptible to complement lysis. To understand how these bacterial pores form in lipid bilayers and the role CD59 plays in complement regulation, we determined the crystal structure of human CD59 bound to ILY. Here, we show the ILY-CD59 complex at 3.5 Å resolution and identify two interfaces mediating this host-pathogen interaction. An ILY-derived peptide based on the binding site inhibits pore formation in a CD59-containing liposome model system. These data provide insight into how CD59 coordinates ILY monomers, nucleating an early prepore state, and suggest a...
Molecular Immunology, 2010
The two N-linked oligosaccharides in native human C9 were deleted by site-specific mutagenesis. This aglycosyl-C9 did not differ from its native form in hemolytic and bactericidal activity. A new N-glycosylation site (K311N/ E313T) was introduced into the turn of a helix-turn-helix [HTH] fold that had been postulated to form a transmembrane hairpin in membrane-bound C9. This glycosylated form of human C9 was as active as the native protein suggesting that the glycan chain remains on the external side of the membrane and that translocation of this hairpin is not required for membrane anchoring. Furthermore, flow cytometry provided evidence for the recognition of membrane-bound C9 on complement-lysed ghosts by an antibody specific for the HTH fold. A new N-glycosylation site (P26N) was also introduced close to the N-terminus of C9 to test whether this region was involved in C9 polymerization, which is thought to be required for cytolytic activity of C9. Again, this glycosylated C9 was as active as native C9 and could be induced to polymerize by heating or incubation with metal ions. The two C-terminal cystines within the MACPF domain could be eliminated partially or completely without affecting the hemolytic activity. Free sulfhydryl groups of unpaired cysteines in such C9 mutants are blocked since they could not be modified with SHspecific reagents. These results are discussed with respect to a recently proposed model that, on the basis of the MACPF structure in C8α, envisions membrane insertion of C9 to resemble the mechanism by which cholesterol-dependent cytolysins enter a membrane.
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...
A mechanism for the insertion of complement component C9 into target membranes
Molecular Immunology, 1986
C9 is a globular serum protein which can insert and polymerise in a target membrane to form a large membrane channel. The ability to insert in the membrane is conferred by amphipathetic elements of secondary structure in the central part of the molecule. Towards each end high cysteine domains are found, one of which is homologous to the apoprotein binding domains of the LDL receptor. From the sequence and topological data for C9 we present a model for its structure and insertion into the membrane.