Date of Award

Summer 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

William F. DeGrado

Abstract

Integrin’s are the principal cell surface receptors that link the cytoskeleton to the extracellular matrix. They exist in active conformations that can bind extracellular ligands and resting conformations that cannot. The platelet integrin αIIbβ3 is a prototypical regulated integrin that is resting on a circulating platelet and becomes activated to adhere the platelet to the vascular endothelium or subendothelial matrix.

The integrin is composed of α and β subunits and each subunit contains a single transmembrane helix that form an α/β heterodimer in the resting state. Additionally, each subunit contains a cytosolic domain that binds signaling proteins that affect the resting-active equilibrium. Activation signals are transduced across the membrane by separating the transmembrane heterodimer.

The structure of the resting integrin αIIbβ3’s transmembrane and cytosolic domains was characterized by molecular modeling and NMR spectroscopy. First, software was developed to model transmembrane helix dimers using experimental mutagenesis results as a modeling restraint. Next, the αIIb/β3 transmembrane heterodimer was modeled and the model was compared to published experimental data and other published models. The model correlated well with experimental findings and converged on the same structure as other top performing models, suggesting this conformation approximates the native interface. The model’s interface includes αIIb residue Met987 and β3 residue Leu712. These residues were mutated to cysteine to crosslink peptides corresponding to the αIIb and β3 cytosolic tails, and the disulfide-linked construct was probed by NMR spectroscopy.

NMR revealed that the αIIb and β3 cytosolic tails have a dynamic interface. The αIIb subunit is natively unstructured and the β3 subunit consists of a hydrophobic helix followed by two amphiphilic helices. The amphiphilic portions of β3 include domains that interact with cytosolic proteins, but the membrane embedding of its hydrophobic faces sequesters some of the interacting residues. This result suggests that the integrin’s resting-active equilibrium is coupled to an equilibrium between membrane embedded and solvent exposed conformations of the β3 cytosolic tail, providing new insight into integrin activation.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS