A. Binding of ligand-mimetic PAC-1 IgM by cysteine-scanning mutants in α_IIb (upper panel) and β₃ (lower panel). Results in absence (Ca) and presence of activation (Mn/PT25-2) are shown, along with wild-type (WT) and GFFKR/GAAKR mutant controls. Leucine-scanning results from a previous study (Luo et al., 2005) are also plotted. B and C. Effects of other mutations in 293T (B) and CHO-K1 (C) transient transfectants.

Disulfide crosslinking in transfectants between subunits with indicated cysteine mutations. As described in Methods, data is with 2-BP, freeze-thaw, and Cu-phenanthroline, except GPA residues 73–80 are with DTT and Cu-phenanthroline. A and B show the same α_IIbβ₃ disulfide crosslinking data, plotted against β₃ (A) or α_IIb sequence position (B). C. GPA TM domain disulfide crosslinking in α_IIbβ₃/GPA71–131 chimera. D and E show the same crosslinking data as in A–C, plotted against Cα-Cα distance. D. Crosslinking efficiency in α_IIbβ₃/GPA71–131 chimera is plotted against Cα-Cα distance in the GPA solid state NMR structure (Smith et al., 2001). E. Crosslinking efficiency in α_IIbβ₃ is plotted against Cα-Cα distance in the final integrin structure. Solid lines show the assumed relationship between crosslinking efficiency and Cα-Cα distance, and dashed lines show the distance, above which, restraint violation was penalized in Membrane Rosetta.

A. Sequences of GPA and α_IIbβ₃ integrin TM and cytoplasmic domains. Numbers in red show positions tested in panels B–E. B–E. Disulfide crosslinking of α_IIbβ₃ with indicated cysteine mutations in 293T transfectants, with or without Cu-phenanthroline, freeze-thaw, and 2-BP treatment as indicated. Immunoprecipitated ³⁵S-labeled material was subjected to nonreducing SDS-PAGE and autoradiography. Positions of αIIb (α), β3 (β) and αIIbβ3 heterodimer (α-β) are shown. F. Integrin αIIbβ3/GPA chimeras. G. Ligand binding by chimeras. Binding of ligand mimetic PAC-1 (IgM) (upper panel) or FITC-labeled fibrinogen (Fg) (lower panel) to 293T transfectants was measured in the presence of 1mM Ca²⁺ or 1mM Mn²⁺ plus 10 μg/ml activating mAb PT25-2. Binding is expressed as mean fluorescence intensity (MFI) of Fg or PAC-1 relative to MFI of Cy3-labled anti- β3 mAb AP3. H. αIIbβ3/GPA60–131 (closed symbols) and αIIbβ3/GPA71–131 (open symbols) chimeras show similar disulfide crosslinking.

A. The disulfide/Rosetta structural ensemble superimposed on the 46-residue TM segments. B. The cluster-center structure. C. The sequences, with residues in crosslinking peaks asterisked. D. Transparent molecular surfaces for α_IIb and β₃ TM segments with all sidechains shown. In A–D, sidechains and Gly Cα spheres in the crosslinking peaks in the 23 residue hydrophobic segment are shown in red or green. Residues in the cytoplasmic juxtamembrane interface region (18 to 12 Å from the membrane center) are cyan. E. A blow-up of the lower TM, JM, and cytoplasmic segments. Hydrogen bonds are dashed green lines. Nitrogens, oxygens, and sulfurs are blue, red and yellow, respectively. F–H. Crick helical-net diagrams (Chothia et al., 1981). Cylindrical helical surfaces are cut at one position along the circumference, unrolled, and aligned at the helical interfaces. I. Superposition of the integrin and GPA TM helices, on residues that form the two ridges forming the groove in β₃ and one GPA monomer in which the GXXXG motifs nestle.

A. Low energy orientations of the α_IIbβ₃ ectodomain on the cell surface. Four favorable cell surface orientations of intact α_IIbβ₃ are shown, superimposed on the TM domains. The ligand-binding α_IIbβ-propeller and β₃ I domains are shown in magenta, and ligand-binding I domain Mg²⁺ ion as an orange sphere. The outer bounds of the hydrophobic, interface, and polar regions of the membrane are shown as black, red, and green lines, respectively. B–D. The NMR bicelle structures of β₃ (Lau et al., 2008b) (B) and α_IIb (Lau et al., 2008a) (D) are shown in the same orientation as the subunits of the α_IIbβ₃ complex (C). E. Superposition on the α_IIbβ₃ complex of the NMR structure of β3 integrin cytoplasmic tail fragment in complex with talin F3 domain (Wegener et al., 2007). The integrin heterodimer structure is in green. The talin F3 domain is in cyan and the β3 cytoplasmic tail fragment of the NMR structure is in red. The side chains of the two phenylalanine residues (F727 and F730) are shown as sticks.

A–F. Effect of restraint omission. Models were generated using different subsets of 12.5, 25, or 50% of the total restraints as described in the Supplement. A, C and E. Models were compared over Cα atoms to the final structure made with all restraints, and in the case of GPA, also to the GPA NMR structure. By definition, models made with 100% of restraints are identical to the final structure. B, D, and F. Models generated with a subset of restraints were scored for violation of the omitted restraints, i.e., those not used in model generation. The per residue RMS distance violation, i.e., distance above the upper bound (see Fig. 2D and E), is shown. How well the models satisfy omitted restraints is a measure of model accuracy similar to the R_free value in crystallography. G. The ensemble of 20 GPA Disulfide/Rosetta structures, showing all heavy atoms (green) superimposed on the solid state NMR structure (red). H. Cartoon of the central GPA Disulfide/Rosetta structure (green) superimposed on the solid state NMR structure (red) with sidechains (or spheres for Gly) shown for residues in crosslinking peaks (asterisked in the sequence insert).