The leukocyte integrin ?X?2 (CD11c/CD18) is an important cell adhesion protein in inflammatory responses of dendritic and myeloid cells. ?X?2 binds to a broad range of ligands through a canonical G-nucleotide-binding domain or inserted (?I)-domain when regulating immune cell migration from vasculature to the site of inflammation. The isolated ?I-domain crystal structures solved to date show that the ?I-domain allosterically changes shape and matures from the closed/low affinity to open/high affinity state (inside-out signaling). It has yet to be determined how the allosteric site and ligand-binding region of the ?I-domain couple and how small molecule antagonists inhibit ligand binding by disrupting allostery. These structural alterations of the ?I-domain on the cell surface have been theorized to control the intracellular recruitment of adaptors that link to signal transduction pathways and cytoskeleton rearrangements and ultimately regulate immune cell emigration when integrins bind ligands on the cell membrane. This proposal builds on previous progress in determining crystal structures of intact ectodomain ?X?2 and ?L?2 and elucidating the roles of secondary structural motions with functional assays. These structures showed that the ?I-domain on the intact integrin platform unprecedentedly undergoes a shape-shifting pathway of allosteric activation that differs from the laid-out ?I-domain activation mechanism but correlates well with the limited number of NMR and MD studies. We hypothesize that the ?I- domain samples into an ensemble of closed/low affinity state conformations and antagonist blocks ligand binding while stabilizing a limited ensemble of closed/low affinity conformations. An NMR strategy established in the laboratory will be employed to characterize in-solution dynamics of the ?I-domain and further analyze the molecular basis of how an antagonist decouples the ligand affinity and allostery.

Public Health Relevance

Leukocyte integrin ?X?2 provides key cellular regulatory mechanism of immune cell migration in defense against infection and autoimmune dysregulations, thus ?X?2 antagonism is a proven modality for controlling inflammation in autoimmune diseases. However, specifically inhibiting integrin functions, such as affinity and allosteric signaling remains a paramount challenge. Characterization of dynamic properties of ?X I-domain activation and antagonist-induced inhibition will define a new paradigm for ?X?2 antagonism studied under conditions that closely mimic the physiological environment and provide important insights into the integrin functions targeted for human clinical intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Small Research Grants (R03)
Project #
5R03AI139651-02
Application #
9694614
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Singleton, Kentner L
Project Start
2018-05-07
Project End
2020-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
2
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Houston
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
036837920
City
Houston
State
TX
Country
United States
Zip Code
77204