Structural Analysis of Engineered Bb Fragment of Complement Factor B Karthe Ponnuraj, Yuanyuan Xu, Kevin Macon, Dwight Moore, John E Volanakis, Sthanam V.L Narayana  Molecular Cell  Volume 14, Issue 1, Pages 17-28 (April 2004) DOI: 10.1016/S1097-2765(04)00160-1

Figure 1 Cartoon Representation of C3-Convertase Formation C3b attached to an activating surface binds factor B, which undergoes conformational changes suitable for the cleavage by factor D, resulting in C3-convertase formation. Molecular Cell 2004 14, 17-28DOI: (10.1016/S1097-2765(04)00160-1)

Figure 2 Structure and Functional Assessment of BbC428-C435 (A) Ribbon representation of BbC428-C435 crystal structure. The serine protease (SP) domain is represented in gold, the vWFA domain in cyan, and the linker joining them in deep blue. The small panel on the right depicts the engineered disulfide bond in the vWFA domain, and the one on left the disulfide bond joining the linker to the SP domain. (B) Three different views of electrostatic surface representation of BbC428-C435. Surface charge distribution was calculated using GRASP (Nicholls et al., 1991). Positively charged regions are represented in blue, negatively charged regions in red, and both polar and nonpolar regions are in white. BbC428-C435 shows three electropositive patches. Region 1 represents the L2 loop of SP domain, and regions 2 and 3 are at the intersection of SP and vWFA domains. (C) Binding of factor B and Bb to CVF. Factor B, factor B plus factor D, wt Bb, or BbC428-C435 was added to microwells precoated with CVF (10 μg/ml) and incubated at 37°C for 2 hr. Bound Bb was detected by rabbit anti-Bb IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG. Molecular Cell 2004 14, 17-28DOI: (10.1016/S1097-2765(04)00160-1)

Figure 3 Structure and Comparison of the vWFA Domain of BbC428-C435 with Integrin I Domain Structures (A) Overall comparison of vWFA domain of BbC428-C435 (cyan) with integrin I domain open conformation structures α2 (yellow) and αM (blue). The two disordered regions of BbC428-C435 are indicated by dotted lines. (B) (Top left) Superposition of α7 helix in ligand-bound/open (yellow) and ligand-free/closed (red) crystal structures of α2 I domain. (Top right) Superposition of Bb-vWFA domain α7 helix (cyan) with the corresponding helix of ligand-bound/open α2 I domain (yellow). (Bottom left) Comparison of MIDAS site residues observed in ligand-bound/open (yellow) and ligand-free/closed (red) crystal structures of α2 I domain. (Bottom right) Superposition of MIDAS site residues of BbC428-C435-vWFA domain (cyan; residue numbers indicated by asterisk) and ligand-bound/open α2 I domain (yellow). For clarity, the water molecules that interact with the metal atom (M) are not included in the bottom left and right panels. (C) Detailed comparison of MIDAS structures of BbC428-C435 (top left), α2 I-collagen complex (top right), αL I–ICAM-1 complex (bottom left), and pseudo-liganded αL I domain (bottom right). The metal atom is shown as a magenta ball. Black solid and dotted lines indicate the metal coordination and hydrogen bonds, respectively. Residue E in the top right and bottom left panels corresponds to the glutamate of collagen and ICAM-1 molecules, respectively. In the bottom right panel residue E corresponds to the neighboring I domain. Molecular Cell 2004 14, 17-28DOI: (10.1016/S1097-2765(04)00160-1)

Figure 4 Structure of BbC428-C435-SP Domain (A) Stereo diagram of BbC428-C435-SP domain showing the surface loops. (B) Superposition of the 668(189)-673(194) segment of BbC428-C435 (gold) with the corresponding regions of active serine proteases trypsin (dark gray, 1TRN), protein C (red, 1AUT), and factor Xa (green, 1FAX). The N-terminal ends of all these molecules (except BbC428-C435) are also shown. (C) Stereo diagram showing the detailed comparison of the C670(191)-G675(196) segment of BbC428-C435 with the corresponding segment of trypsin. The boxed area indicates the orientational difference of the carbonyl oxygen of residue 671(192). (D) Stereo diagram showing the superposition of S1 pocket of BbC428-C435 and trypsin. Molecular Cell 2004 14, 17-28DOI: (10.1016/S1097-2765(04)00160-1)

Figure 5 Structures of BbC428-C435 Inhibitor Complexes (A) Superposition of the active sites of BbC428-C435 (gold) and BbC428-C435-BCX583 (light green) crystal structures. The inhibitor (BCX583) covalently linked to Ser195 is held in position by hydrogen bonding to Asp715(226) through a water molecule (lavender). The L2 loop (red) is seen only in the inhibitor complex structure. (B) Comparison of the active sites of BbC428-C435-DIP and BbC428-C435. The backbone of the inhibitor-bound BbC428-C435 is shown in light green color, while the inhibitor-free BbC428-C435 is shown in gold color. Covalently bound DIP molecule points its phosphoryl oxygen into the putative oxyanion hole (marked with black star) and forces it to acquire a tight β turn from a zymogen-like 310 helix (transparent red color) conformation. (C) Stereo close-up of the superposition of the 670(191)-674(194) segment of BbC428-C435 (gold) and BbC428-C435-DIP complex (light green). The orientational difference of carbonyl oxygen of the residue 671(192) is indicated in the boxed area. Molecular Cell 2004 14, 17-28DOI: (10.1016/S1097-2765(04)00160-1)