Integrins ?IIb?3 and ?5?1 are cell adhesion receptors that bind extracellular ligands, transduce signals bidirectionally across plasma membranes, and play important roles in the vasculature. Ligand binding and integrin activation are coupled to two distinct, large conformational changes, extension at the integrin knees and opening of the ligand-binding headpiece. On cell surfaces, integrins dynamically equilibrate between two low affinity conformations, bent-closed and extended-closed, and a high affinity extended-open conformation. Investigating integrin conformational equilibria, which have never been measured for any integrin, is paramount for understanding how the conformational ensemble dictates the functional output of integrins. Furthermore, how ligands bind integrins and drive headpiece opening remain incompletely understood.
Aim 1 continues work on integrin ?IIb?3, which recognizes an Arg-Gly-Asp (RGD) motif. We characterize binding of an AGDV peptide from fibrinogen, which lacks the Arg of RGD, and demonstrate that its engagement of the MIDAS in the ?-subunit is sufficient to open the integrin headpiece. We also complete work on the structural basis for quinine-dependent antibody binding to platelet integrin ?IIb?3, which causes drug-induced immune thrombocytopenia (DITP).
Aim 2 focuses on another RGD-binding integrin, ?5?1, which binds its primary ligand fibronectin (Fn) and directs its assembly into the extracellular matrix. Their interaction is important for angiogenesis, vascular development, and cancer progression. Intriguingly, ?5?1 also binds the non-RGD ligand Invasin (Inv) to mediate bacterial internalization of Yersenia spp. that cause plague and gastroenteritis. We characterize the conformational states of ?5?1 ectodomain by negative stain electron microscopy. We examine complexes of ?5?1 with function-perturbing Fabs and Inv to define their binding sites and the integrin conformations they stabilize.
Aim 3 investigates crystal structures of the ?5?1 headpiece and its complexes with Fn and Inv fragments. Structures will illustrate how RGD in Fn3 module 10 and the synergy site in Fn3 module 9 bind ?5?1 and the extent to which the non-RGD ligand Inv mimics Fn binding, and provide insight into how binding of different ligands affect headpiece opening.
Aim 4 measures conformational equilibria for ?5?1, and how conformational equilibria mix with the intrinsic affinity of a specific integrin conformation for ligand to yield the apparent affinity measured for an integrin ectodomain fragment or an intact integrin on the cell surface. For the first time, the conformational equilibria for integrin extenson and headpiece opening will be separately measured, and related to regulation of affinity for fibronectin. The effects of transmembrane domain association, glycosylation state, and oncogenic cell transformation on conformational equilibria of ?5?1 are also studied. Results from our work will guide the design of higher affinity and novel non-RGD based ?5?1-inhibitors as therapeutics for pathological angiogenesis and cancer.

Public Health Relevance

The cell surface protein, integrin ?5?1, directs formation of new blood vessels during normal development of the vascular system and in tumors, and has also been implicated in mediating infections by bacteria that cause gastroenteritis and plague. This grant will explore the structure of integrin ?5?1 and how its binding partners, fibronectin and bacterial invasin, alter its shape. Insights obtained from this grant will be critical for developing more effective drugs to treat diseases that depend on the formation of new blood vessels, such as cancer.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL131729-03
Application #
9471837
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Kindzelski, Andrei L
Project Start
2016-04-20
Project End
2020-03-31
Budget Start
2018-04-01
Budget End
2019-03-31
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Boston Children's Hospital
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
Li, Jing; Springer, Timothy A (2018) Energy landscape differences among integrins establish the framework for understanding activation. J Cell Biol 217:397-412
Li, Jing; Su, Yang; Xia, Wei et al. (2017) Conformational equilibria and intrinsic affinities define integrin activation. EMBO J 36:629-645
Li, Jing; Springer, Timothy A (2017) Integrin extension enables ultrasensitive regulation by cytoskeletal force. Proc Natl Acad Sci U S A 114:4685-4690
Su, Yang; Xia, Wei; Li, Jing et al. (2016) Relating conformation to function in integrin ?5?1. Proc Natl Acad Sci U S A 113:E3872-81
Lin, Fu-Yang; Zhu, Jianghai; Eng, Edward T et al. (2016) ?-Subunit Binding Is Sufficient for Ligands to Open the Integrin ?IIb?3 Headpiece. J Biol Chem 291:4537-46
Zhu, Jianghai; Zhu, Jieqing; Bougie, Daniel W et al. (2015) Structural basis for quinine-dependent antibody binding to platelet integrin ?IIb?3. Blood 126:2138-45