NMR Structure of a Disulfide-Crosslinked IIb3 Cytoplasmic Domain Heterodimer
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AbstractThe platelet integrin IIb3 is the prototypic example of regulated integrin function. Thus, IIb3 is present in a resting conformation on unstimulated platelets, but switches to an active conformation following platelet stimulation. Recent experiments suggest that disrupting a heteromeric interaction between the IIb and 3 transmembrane (TM) and cytoplasmic domains shifts IIb3 from its resting to its active conformation. However, structural information about the heteromeric interaction is sparse. Thus far, the structure of the TM heterodimer has only been studied by molecular modeling. Interactions between soluble cytosolic tail peptides have been studied by NMR spectroscopy, but these studies may not reflect native contacts because they fail to account for constraining TM domain interactions. To obtain an NMR structure for the IIb3 cytosolic tail heterodimer that reflects its native structure, we expressed 13C- and 15N-labeled peptides corresponding to IIb residues 9881008 and 3 residues 713762 in E. coli. Residues 987 in IIb and 712 in 3 were replaced with cysteines, based on modeling that predicts the resultant disulfide bond will fix the peptides in their native orientation. Crosslinked heterodimers were dissolved in dodecylphosphocholine micelles at pH 6.5 and analyzed at 37C on a 750 MHz NMR spectrometer. Previously, we presented a preliminary analysis of this construct indicating that when constrained by the proximal disulfide bond, the IIb and 3 cytoplasmic tails interact and the cytosolic tail of 3 consists of three helices. We have now solved the final structure which defines the 3 interface that interacts with the IIb cytoplasmic tail. The IIb-3 heterodimer interface is dynamic, but can be localized to 3 residues 716 and 719 because they have different chemical shifts in the crosslinked heterodimer than they do in the component monomers. This positions 3 residue 723 at the IIb-3 interface, consistent with the putative Arg995-Asp723 salt bridge. Interestingly, the IIb tail is natively unstructured so a static interface for IIb could not be identified. Additionally, the completed structure defines the relative orientations of the three 3 helices. The 3 cytoplasmic tail contains a sharp kink at residue 724 that fixes the membrane embedded helix (residues 713723) and the first cytoplasmic helix (residues 725736) at a right angle. The kink was defined by multiple NMR parameters including NOE distance restraints between residues 721 and 727. The distal cytoplasmic helix (residues 746757) is related to the rest of the molecule by a flexible loop (residues 737 745). N15 NOESY-HSQC crosspeak intensities provide evidence that the flexible loop and distal helix undergo increased motion relative to the first two helices, and the final structure reflects this motion because there is no preferred orientation for the distal helix relative to the first two helices. Lastly, the distal helix and flexible loop are joined by 3s canonical NPXY motif which forms an N-terminal cap for the distal helix. In conclusion, we have solved the NMR structure of a disulfide-crosslinked IIb/3 cytoplasmic tail heterodimer. Our analysis indicates that, when constrained by a disulfide bond, the IIb and 3 cytoplasmic tails interact, providing one mechanism for maintaining IIb3 in a resting state.
