Identification of Stalk Mutations That Stabilize the High Affinity Conformation of αIIbβ3 by Negative Design.
Authors: Donald Jason E. , Zhu Hua , DeGrado William F. , Bennett Joel S. 
Published: October 2019

Abstract 4017

Poster Board III-953

The integrin αIIbβ3 plays an essential role in platelet aggregation. Following platelet stimulation by agonists, αIIbβ3 on the platelet surface shifts from an inactive low affinity conformation unable to bind ligands to an active high affinity conformation that can bind fibrinogen and von Willebrand factor. These αIIbβ3-bound macromolecules then crosslink platelets into aggregates. αIIbβ3 is thought to be present on the platelet surface in a bent conformation that is maintained by interactions involving residues located in its cytoplasmic, transmembrane, and stalk domains. Crystal structures of αIIbβ3 and related integrin extracellular domains indicate that large conformational movements occur during activation and specific point mutations in the transmembrane domain and cytoplasmic domains lead to ligand binding, likely by disrupting the inactive state heterodimer interface. One region of αIIbβ3 that has not been studied in detail is the large intramolecular interface located in the stalk region. To test the hypothesis that the interface between the αIIb and β3 stalks observed in the crystal structure of the αIIbβ3 extracellular domain is an important part of the αIIbβ3 activation equilibrium, we employed a negative design strategy in which the Rosetta energy function was used to predict alanine mutations in β3 that would most destabilize the stalk interface. Negative design is a way to control the direction of protein interactions by introducing changes in protein structure that destabilize undesired interactions. In this case, the undesired interaction is the inactive heterodimer stalk interface. The Rosetta energy function used here has been shown to accurately predict the effect of alanine mutations at protein interfaces. Initially, mutations were predicted based on the available crystal structure of the αvβ3 heterodimer. Subsequently, a crystal structure for inactive αIIbβ3 was reported. The functional consequences of the predicted mutations were then tested by introducing the mutations into full-length β3 expressed in CHO cells. Two mutations, V664A and Y594A, were selected by the design algorithm as particularly disruptive in the interface and caused substantial constitutive αIIbβ3 activation when introduced into full-length β3. By contrast, the mutation, D552A, predicted to be slightly destabilizing in αVβ3, but not destabilizing in αIIbβ3, had essentially no effect on αIIbβ3 activity. These results confirm the utility of the negative design approach to identifying functionally significant regions in integrins. Further, they demonstrate that the stalk interface, much like the transmembrane domain interface, plays an important role in the equilibrium between active and inactive states of αIIbβ3. The large intermolecular stalk interface appears to be important for inside-out integrin signaling and deserves further study for its role in the transmission of signals from the intracelluar to the extracellular regions of integrins.

Disclosures:

No relevant conflicts of interest to declare.