Expression in Escherichia coli, Folding in Vitro, and Characterization of the Carbohydrate Recognition Domain of the Natural Killer Cell Receptor NKR-P1A

NKR-P1A is a homodimeric type II transmembrane protein of the C-type lectin family found on natural killer (NK) cells and NK-like T cells and is an activator of cytotoxicity. Toward structure determination by NMR, the recombinant carbohydrate-recognition domain (CRD) of NKR-P1A has been expressed in high-yield in Escherichia coli and folded in vitro. The purified protein behaves as a monomer in size-exclusion chromatography and is bound by the conformation-sensitive antibody, 3.2.3, indicating a folded structure. A polypeptide tag at the N-terminus is selectively cleaved from the CRD after limited trypsin digestion in further support of a compact folded structure. The disulfide bonds have been identified by peptide mapping and electrospray mass spectrometry. These are characteristic of a long form CRD. The 1D NMR spectrum of the unlabeled CRD and the 2D HSQC spectrum of the 15N-labeled CRD are those of a folded protein. Chemical shifts of Hα and NH protons indicate a considerable amount of β-strand structure. Successful folding in the absence of Ca2+, coupled with the lack of chemical shift changes upon addition of Ca2+, suggests that the NKR-P1A-CRD may not be a Ca2+-binding protein.