Refolding and NMR structures of CD4-Lck and CD8alpha-Lck complexes: A novel zinc-mediated dimerization motif

CD4 and CD8 are glycoprotein receptors expressed on the surface of T lymphocytes. CD4 is a monomer and CD8 is a dimer of either two CD8alpha subunits or one CD8alpha and one CD8beta subunit. The cytoplasmic tails of CD4 and CD8alpha interact with the N-terminus of p56lck (Lck), a tyrosine kinase. These interactions are important for T-cell development and activation. To better understand their role in the immune system, the interactions of these components have been reconstituted in vitro and studied biophysically.; CD4 cytoplasmic tail, CD8alpha cytoplasmic tail and Lck N-terminus were produced using an E. coli expression system, and purified under denaturing conditions. CD4-Lck and CD8alpha-Lck complexes were refolded by combining the peptides with Zn2+ in an equimolar ratio. The CD8alpha-Lck complex was purified by gel filtration and its analysis demonstrated CD8alpha, Lck and Zn2+ to be in an approximately 1:1:1 molar ratio. Both CD4-Lck and CD8alpha-Lck complexes were shown to have unstructured residues by heteronuclear single quantum coherence (HSQC) spectra. The number of unstructured residues was reduced by limited proteolysis of the complexes and identification of products by mass spectrometry.; Structures for both complexes were determined by nuclear magnetic resonance (NMR) with a backbone root mean square deviation (RMSD) of ∼0.4 A for structured residues. The structures show that Zn2+ is coordinated by four cysteines in both complexes, two from a CXC sequence in CD4 or CD8alpha, and two from a CXXC sequence in Lck. In the CD4-Lck complex, residues Asp 11-Leu 14 of Lck form an alpha-helix, followed by a beta-hairpin that positions Cys 20 and Cys 23 for Zn2+ coordination. Residues Arg 406-Ser 415 of CD4 form an amphipathic helix which packs with the helical region in Lck. The helix is followed by a loop that drapes across the Lck beta-hairpin. Cys 420 and Cys 422 in this loop complete tetrahedral Zn2+ coordination. The CD8alpha-Lck complex is similar but lacks the helices found in the CD4-Lck complex.; In addition to Zn2+ coordination, both complexes are stabilized by specific hydrophobic interactions which are more numerous in CD4-Lck than in the smaller CD8alpha-Lck complex. The CD4-Lck and CD8alpha-Lck interactions are tight, and their affinities were measured by isothermal titration calorimetry (ITC). The binding affinity of CD4 for Lck was 0.40 muM, and the affinity of CD8a for Lck was 0.90 muM. A question arising from the CD4-Lck and CD8alpha-Lck structures is whether the interactions affect another (eg. SH3, SH2 or kinase) domain of Lck.