Natural killer (NK) cells play a recently recognized and critical role in innate immunity, functioning through an array of stimulatory and inhibitory cell-surface receptors that mediate recognition and signaling events. The major histocompatibility complex (MHC) class I homolog MIC-A is a stress-inducible self antigen that is broadly recognized by NK cells, CD8+ alpha beta T cells and the V51-bearing subset of gamma/delta T cells independent of beta2-microglobulin and bound peptides. Consequently, MIC-A has been proposed to """"""""play an important role in the surveillance of transformed, infected and damaged cells"""""""" by the innate immune system in intestinal epithelium and epithelially derived tumors. MIC-A recognition by these cells is mediated through interactions with the stimulatory NK receptor NKG2D, a divergent member of the C-type lectin-like protein family and a distant relative of other members of the NKG2 family of NK cell receptors and CD94. Our recent crystallographic analysis of the three-dimensional structure of MIC-A reveals a dramatically altered MHC class I fold, both in detail and overall domain organization. We propose to characterize the biochemical and structural details of the interaction between the NK cell receptor NKG2D and the MIC family of proteins. This knowledge will allow us to understand an important NK recognition event, mediated by NKG2D, whose target is clearly unlike that of alpha beta T cell receptors or other NK cell receptors which recognize more conventional MHC class I proteins and homologs. We will achieve this goal by 1) expressing soluble forms of the extracellular domains of a series of members of the MIC protein family and NKG2D, using multiple expression systems; 2) determining binding affinities by both qualitative and quantitative assays; 3) delineating a proposed interaction site on MIC-A by site-directed mutational analysis; and 4) crystallizing and determining the three-dimensional structures of individual proteins and appropriate complexes by x-ray crystallography. We will also 5) determine the effect glycosylation has on i) the MIC/NKG2D interaction; ii) the stability of these proteins; and iii) their structures. Since the initial submission of this application, we have crystallized three of the species we propose to study, including the MIC-AfNKG2D complex, and have collected preliminary SPR binding data for one interaction (baculovirus-expressed MIC-A and bacterially-expressed NKG2D).

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Rathbun, Gary
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Fred Hutchinson Cancer Research Center
United States
Zip Code
Finton, Kathryn A; Strong, Roland K (2012) Structural insights into activation of antiviral NK cell responses. Immunol Rev 250:239-57
Xu, Bin; Pizarro, Juan C; Holmes, Margaret A et al. (2011) Crystal structure of a gammadelta T-cell receptor specific for the human MHC class I homolog MICA. Proc Natl Acad Sci U S A 108:2414-9
Correnti, Colin; Clifton, Matthew C; Abergel, Rebecca J et al. (2011) Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities. Structure 19:1796-806
Bandaranayake, Ashok D; Correnti, Colin; Ryu, Byoung Y et al. (2011) Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res 39:e143
Kaiser, Brett K; Pizarro, Juan Carlos; Kerns, Julie et al. (2008) Structural basis for NKG2A/CD94 recognition of HLA-E. Proc Natl Acad Sci U S A 105:6696-701
McBeth, Christine; Seamons, Audrey; Pizarro, Juan C et al. (2008) A new twist in TCR diversity revealed by a forbidden alphabeta TCR. J Mol Biol 375:1306-19
Vigdorovich, Vladimir; Miller, A Dusty; Strong, Roland K (2007) Ability of hyaluronidase 2 to degrade extracellular hyaluronan is not required for its function as a receptor for jaagsiekte sheep retrovirus. J Virol 81:3124-9
Andersen-Nissen, Erica; Smith, Kelly D; Bonneau, Richard et al. (2007) A conserved surface on Toll-like receptor 5 recognizes bacterial flagellin. J Exp Med 204:393-403
Holmes, Margaret A; Paulsene, Wendy; Jide, Xu et al. (2005) Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration. Structure 13:29-41
Kaiser, Brett K; Barahmand-Pour, Fariba; Paulsene, Wendy et al. (2005) Interactions between NKG2x immunoreceptors and HLA-E ligands display overlapping affinities and thermodynamics. J Immunol 174:2878-84

Showing the most recent 10 out of 18 publications