A combined biochemical and cellular approach reveals Zn2+-dependent hetero- and homodimeric CD4 and Lck assemblies in T cells

The CD4 or CD8 co-receptors’ interaction with the protein-tyrosine kinase Lck is widely accepted as the initiator of the tyrosine phosphorylation cascade leading to T-cell activation. These co-receptors potentially enhance T-cell antigen sensitivity, but how they function is still debated. A critical question is: to what extent are co-receptors and signal-initiating Lck coupled? Our contribution concerns the small – but indispensable for CD4- and CD8-Lck formation – element Zn2+. The intracellular Zn2+ pool is strictly buffered but undergoes dynamic changes, also reported during T-cell activation. Furthermore, the identical Zn2+-binding cysteinyl residues may alter co-receptor dimerization or heterodimerization with Lck. Following initial research demonstrating a significant difference in the affinity of Zn2+ to CD4 and CD4-Lck in solution, we combined biochemical and cellular approaches to show that fluctuations of buffered Zn2+ in physiological ranges indeed influence Zn(CD4)2 and Zn(CD4)(Lck). This conclusion was supported by the simulation of complexes’ equilibria, demonstrating that Zn2+ changes can alter the molar ratio between those complexes. In T cells, increased intracellular free Zn2+ concentration causes higher CD4 partitioning in the plasma membrane by a still unknown mechanism. We additionally found that CD4 palmitoylation decreases the specificity of CD4-Lck formation in the reconstituted membrane model, suggesting that this reversible modification may also be involved. Our findings help elucidate co-receptor-Lck coupling stoichiometry and demonstrate that intracellular free Zn2+ has a major role in the interplay between CD4 dimers and CD4-Lck assembly.