soluble mac  (SMAC Corp)

Journal: Cell Discovery

Article Title: Potent and broadly neutralizing antibodies against sarbecoviruses induced by sequential COVID-19 vaccination

doi: 10.1038/s41421-024-00648-1

Figure Lengend Snippet: a Cryo-EM structures of the BA.1 S trimer in complex with the antibody PW5-570 IgG. b Structure of BA.1-RBD–PW5-570. The RBD is displayed in tomato surface mode. The heavy chain and light chain of PW5-570 are shown as ribbons colored in dark green and light green, respectively. c Close-up view of the PW5-570 epitope on the RBD. ACE2-binding epitopes are represented by yellow dashed lines. d Detailed interactions between the PW5-570 and BA.1 S RBD. Hydrogen bonds are represented by dashed lines. Hydrophobic interactions are marked with red circles. e PW5-570 clash with the ACE2. ACE2 is marked yellow.

Article Snippet: The mammalian expression plasmid for dimeric soluble ACE2 was purchased from Addgene.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Cell Discovery

Article Title: Potent and broadly neutralizing antibodies against sarbecoviruses induced by sequential COVID-19 vaccination

doi: 10.1038/s41421-024-00648-1

Figure Lengend Snippet: a Cryo-EM structure of the XBB S in complex with the antibody PW5-535 IgG. b Cryo-EM structures of the SARS-CoV S in complex with the antibody PW5-535 IgG. c Structure of XBB RBD–PW5-535. The RBD is displayed in tomato. The heavy chain and light chain of PW5-535 are shown as ribbons colored in dark purple and light purple, respectively. d Close-up view of the PW5-535 epitope on the XBB RBD and SARS-CoV RBD. e Detailed interactions between the PW5-535 and XBB S RBD. f PW5-535 causes steric hindrance with ACE2. g Detailed interactions between the PW5-535 and SARS-CoV S RBD and SD1. Hydrogen bonds are represented by dashed lines. Hydrophobic interactions are marked with red circles.

Article Snippet: The mammalian expression plasmid for dimeric soluble ACE2 was purchased from Addgene.

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural basis for membrane attack complex inhibition by CD59

doi: 10.1038/s41467-023-36441-z

Figure Lengend Snippet: The MAC pore is formed from the sequential and stepwise assembly of complement proteins: C5b, grey; C6, blue; C7, orange; C8α, pink; C8β, magenta; C8γ, light purple; C9, alternating monomers are yellow and green. During assembly, complement proteins undergo dramatic structural re-arrangements in which two helical bundles within their MACPF domains unfurl into membrane-inserting β-hairpins. CD59 (cyan) binds at two stages of this assembly process (C5b8 and C5b9) to block membrane perforation and C9 polymerization. Images are rendered from structural models. C5b6 and all MAC assemblies: PDB ID: 6H03 ; Soluble forms of complement proteins are derived from C6: PDB ID: 3T5O ; C8: PDB ID: 3OJY ; C9: PDB ID: 6CXO . C7 was derived from an AlphaFold2 prediction: AlphaFold Protein Structure Database P10643 .

Article Snippet: Here, only TMH1 has unfurled while TMH2 remains helical (Fig. ), similar to the terminal C9 of the soluble inhibited MAC (sMAC) complex .

Techniques: Membrane, Blocking Assay, Derivative Assay

Journal: Nature Communications

Article Title: Structural basis for membrane attack complex inhibition by CD59

doi: 10.1038/s41467-023-36441-z

Figure Lengend Snippet: A , B Cholesterol depletion assays. A representative image (out of 10 randomly selected locations) for each condition is shown. Scale bars, 50 μm. A Complement was activated on CHO cells with a polyclonal anti-CHO IgG antibody. Cells were incubated with C9-depleted human serum supplemented with a chemically labeled fluorescent C9 (C9-Alexafluor 568) capable of forming MAC. B CHO cells treated with MβCD to deplete cholesterol. Complement activation and C9 detection is as described in ( A ). Insets show a zoomed in view of single cell. Cartoon schematics highlight the pattern of MAC deposition. C CryoEM map of the C5b9 2 -CD59 complex in a lipid nanodisc applying a positive B-factor of +50 Å 2 (transparent surface). Surface rendering of the protein model is underlayed. CD59 is cyan, C8β is magenta and the remaining complement proteins are grey. D Map generated from the coarse-grained model of the C5b8-CD59 complex including the GPI anchor (green). Protein components colored as in panel ( C ). Water molecules in proximity to the membrane are shown as blue spheres. Initial and final configurations for the three MD repeats are included in the Supplementary Data Files.

Article Snippet: Here, only TMH1 has unfurled while TMH2 remains helical (Fig. ), similar to the terminal C9 of the soluble inhibited MAC (sMAC) complex .

Techniques: Incubation, Labeling, Activation Assay, Generated, Membrane

Journal: Nature Communications

Article Title: Structural basis for membrane attack complex inhibition by CD59

doi: 10.1038/s41467-023-36441-z

Figure Lengend Snippet: A MAC assembles on cholesterol-rich microdomains (red hexagons) in the plasma membrane. While C7 anchors the growing MAC, C8β thins the bilayer and primes the membrane for rupture by C8α (pink). C9 joins the assembly, undergoing discrete structural transitions to form the pore. The MACPF domain of soluble C9 makes an initial contact and aligns the central β-sheet. The pore-forming β-hairpins extend sequentially. TMH1 is followed by TMH2, which allow C9 polymerization to complete the MAC pore. B CD59 is a GPI-anchored cell surface receptor that clusters in cholesterol-rich microdomains. Upon complement activation, CD59 could respond to membrane thinning by C8β and capture C8α as its transmembrane β-hairpins are extending (pink). While bound to C8α, CD59 is positioned to deflect the cascading C9 β-hairpins and divert their membrane trajectory (green). The next C9 molecule (yellow) is able to bind but trapped in an intermediate conformation in which TMH2 remains helical (red) and in which C9 polymerization is halted.

Article Snippet: Here, only TMH1 has unfurled while TMH2 remains helical (Fig. ), similar to the terminal C9 of the soluble inhibited MAC (sMAC) complex .

Techniques: Clinical Proteomics, Membrane, Cell Surface Receptor Assay, Activation Assay

Journal: Nature Communications

Article Title: Structural basis for membrane attack complex inhibition by CD59

doi: 10.1038/s41467-023-36441-z

Figure Lengend Snippet: The MAC pore is formed from the sequential and stepwise assembly of complement proteins: C5b, grey; C6, blue; C7, orange; C8α, pink; C8β, magenta; C8γ, light purple; C9, alternating monomers are yellow and green. During assembly, complement proteins undergo dramatic structural re-arrangements in which two helical bundles within their MACPF domains unfurl into membrane-inserting β-hairpins. CD59 (cyan) binds at two stages of this assembly process (C5b8 and C5b9) to block membrane perforation and C9 polymerization. Images are rendered from structural models. C5b6 and all MAC assemblies: PDB ID: 6H03 ; Soluble forms of complement proteins are derived from C6: PDB ID: 3T5O ; C8: PDB ID: 3OJY ; C9: PDB ID: 6CXO . C7 was derived from an AlphaFold2 prediction: AlphaFold Protein Structure Database P10643 .

Article Snippet: Initial models for complement proteins were built using coordinates for the soluble MAC structure (sMAC) and the CD59 crystal structure .

Techniques: Membrane, Blocking Assay, Derivative Assay

Journal: Nature Communications

Article Title: Structural basis for membrane attack complex inhibition by CD59

doi: 10.1038/s41467-023-36441-z

Figure Lengend Snippet: A , B Cholesterol depletion assays. A representative image (out of 10 randomly selected locations) for each condition is shown. Scale bars, 50 μm. A Complement was activated on CHO cells with a polyclonal anti-CHO IgG antibody. Cells were incubated with C9-depleted human serum supplemented with a chemically labeled fluorescent C9 (C9-Alexafluor 568) capable of forming MAC. B CHO cells treated with MβCD to deplete cholesterol. Complement activation and C9 detection is as described in ( A ). Insets show a zoomed in view of single cell. Cartoon schematics highlight the pattern of MAC deposition. C CryoEM map of the C5b9 2 -CD59 complex in a lipid nanodisc applying a positive B-factor of +50 Å 2 (transparent surface). Surface rendering of the protein model is underlayed. CD59 is cyan, C8β is magenta and the remaining complement proteins are grey. D Map generated from the coarse-grained model of the C5b8-CD59 complex including the GPI anchor (green). Protein components colored as in panel ( C ). Water molecules in proximity to the membrane are shown as blue spheres. Initial and final configurations for the three MD repeats are included in the Supplementary Data Files.

Article Snippet: Initial models for complement proteins were built using coordinates for the soluble MAC structure (sMAC) and the CD59 crystal structure .

Techniques: Incubation, Labeling, Activation Assay, Generated, Membrane

Journal: Nature Communications

Article Title: Structural basis for membrane attack complex inhibition by CD59

doi: 10.1038/s41467-023-36441-z

Figure Lengend Snippet: A MAC assembles on cholesterol-rich microdomains (red hexagons) in the plasma membrane. While C7 anchors the growing MAC, C8β thins the bilayer and primes the membrane for rupture by C8α (pink). C9 joins the assembly, undergoing discrete structural transitions to form the pore. The MACPF domain of soluble C9 makes an initial contact and aligns the central β-sheet. The pore-forming β-hairpins extend sequentially. TMH1 is followed by TMH2, which allow C9 polymerization to complete the MAC pore. B CD59 is a GPI-anchored cell surface receptor that clusters in cholesterol-rich microdomains. Upon complement activation, CD59 could respond to membrane thinning by C8β and capture C8α as its transmembrane β-hairpins are extending (pink). While bound to C8α, CD59 is positioned to deflect the cascading C9 β-hairpins and divert their membrane trajectory (green). The next C9 molecule (yellow) is able to bind but trapped in an intermediate conformation in which TMH2 remains helical (red) and in which C9 polymerization is halted.

Article Snippet: Initial models for complement proteins were built using coordinates for the soluble MAC structure (sMAC) and the CD59 crystal structure .

Techniques: Clinical Proteomics, Membrane, Cell Surface Receptor Assay, Activation Assay