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, 405 (2), 325-30

Structure of Human Complement C8, a Precursor to Membrane Attack

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Structure of Human Complement C8, a Precursor to Membrane Attack

Doryen Bubeck et al. J Mol Biol.

Abstract

Complement component C8 plays a pivotal role in the formation of the membrane attack complex (MAC), an important antibacterial immune effector. C8 initiates membrane penetration and coordinates MAC pore formation. High-resolution structures of C8 subunits have provided some insight into the function of the C8 heterotrimer; however, there is no structural information describing how the intersubunit organization facilitates MAC assembly. We have determined the structure of C8 by electron microscopy and fitted the C8α-MACPF (membrane attack complex/perforin)-C8γ co-crystal structure and a homology model for C8β-MACPF into the density. Here, we demonstrate that both the C8γ protrusion and the C8α-MACPF region that inserts into the membrane upon activation are accessible.

Figures

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Fig. 1
Fig. 1
Two-dimensional images of negatively stained C8. C8 (2.5 μl of 0.03 mg/ml) was applied to a carbon-coated copper-palladium grid, glow-discharged for 10 s at 20 mA. Grids were negatively stained with 0.75% uranyl formate using the two-drop method. Images were taken under low-dose conditions (∼10 e2 per exposure) at a magnification of 59,000× on a Tecnai F30 microscope. Micrographs were digitized using a SCAI scanner (Z/I Imaging) at a step size of 7 μm and binned by a factor of 4, resulting in a pixel size of 4.74 Å/pixel (a). (b) 5167 windowed particles were subjected to 10 cycles of reference-free alignment using EMAN and classified into 362 classes. Representative 2D class averages indicate a wide range of orientations. The scale bar represents 110 Å.
Fig. 2
Fig. 2
Three-dimensional reconstruction of C8 at  24-Å resolution. A Gaussian blob was used as reference to initiate the angular assignment of raw images using EMAN. The refinement was iterated until convergence, and a surface rendering of the final reconstruction is visualized from three views differing by a 90° rotation (a). Angles and axes of rotation are indicated by arrows. C8 can be described as having a pseudo-2-fold symmetric globular domain flanked by a protrusion. The scale bar represents 45 Å. Maps were visualized using CHIMERA. (b) Two-dimensional projections of the reconstruction and corresponding class averages are depicted in alternating panels, starting with the projection. The scale bar represents 110 Å.
Fig. 3
Fig. 3
Pseudo-atomic modeling of C8. (a) The crystal structure (PDB ID: 2RD7) of the complex between C8α-MACPF (blue ribbons) and C8γ (yellow ribbons) was fit into the reconstruction (grey mesh). Using the C8α-MACPF, a homology model for C8β-MACPF (45% sequence similarity) was built with MODELLER; however, loops larger than 5 residues extending from the core domain were removed. Two possible placements of C8β-MACPF, related by a 180° rotation interchanging the arms of the “L,” were scored. The one shown here corresponds to the lower refinement residual and is rendered as an isosurface filtered to 25 Å (green). Positions were refined using Chimera and PHENIX as two movable rigid bodies (see Table S1 for details). Refined models were scored against the electron microscopy map using a real space correlation coefficient computed using the BSOFT package. Access to the putative transmembrane hairpin of C8α, TMH1 (brown), and the CD59 binding site located in the TMH2 region (tan) is located on the outer face of C8. The figure was prepared using PYMOL. The N and C termini of C8α-MACPF are cyan spheres; the N and C termini of C8β-MACPF are brown spheres. Quaternary structure schematics are shown for each view. Domains are color coded similarly. The scale bar represents 45 Å. (b) Cartoon representation of the model color coded as in panel (a).

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