Homodimerization and Heterodimerization of Minimal Zinc(II)-Binding-Domain Peptides of T-Cell Proteins CD4, CD8α, and Lck

Abstract

Metal-mediated protein oligomerization is an emerging mode of protein-protein interaction. The C-terminal cytosolic domains of T-cell coreceptors CD4 and CD8α form zinc-bridged heterodimers with the N-terminal region of the kinase Lck, with each protein contributing two cysteinate ligands to the complex. Using size exclusion chromatography, 1H NMR, and UV/visible absorption spectroscopy with cobalt(II) as a spectroscopic probe, we demonstrate that small peptides derived from these regions form metal-bridged heterodimers but also homodimers, in contrast to previous reports. The Lck-CD4 and Lck-CD8α cobalt(II)-bridged heterodimer complexes are more stable than the corresponding (Lck)2cobalt(II) complex by factors of 11 ± 4 and 22 ± 9, respectively. These studies were aided by the discovery that cobalt(II) complexes with a cobalt(II)(-Cys-X-X-Cys-)(-Cys-X-Cys-) chromophore show unusual optical spectra with one component of the visible d to d (4A2 to 4T1(P)) transition red-shifted and well separated from the other components. These results provide insights into the basis of specificity of metal-bridged complex formation and on the potential biological significance of metal-bridged homodimers in T-cells.

Metal-coupled folding as the driving force for the extreme stability of Rad50 zinc hook dimer assembly

“…However, these values were measured in the presence of excess Zn 2+ , and thus the metal concentration factor was neglected in the calculation of the stability constant. In the following study, the authors considered both the metal and peptide concentrations and measured the apparent stability constants of Co 2+ -mediated dimers formed by minimal peptides from CD4, CD8α and Lck reporting values, which expressed as apparent formation constants (log K ) are 7.8 for the CD4-Co 2+ -Lck complex and 8.1 for the CD8α-Co 2+ -Lck complex52.…”

Metal-Mediated Affinity and Orientation Specificity in a Computationally Designed Protein Homodimer

“…Zinc has a well-established structural role in protein tertiary and quaternary structure of naturally occurring proteins 17,21–23 , and engineering zinc binding sites was one of the earliest goals in computational protein design. Regan and co-workers and Hellinga and co-workers designed metal-binding sites in proteins twenty years ago 24–27 .…”

Zinc clasp-based reversible toolset for selective metal-mediated protein heterodimerization

“…28 Biophysical studies performed to date show that short model peptides from Lck and CD4 tend to form both homo-and heterodimeric species typically for short CXXC-containing motifs. 21,29 Therefore we rationally optimized the CD4 cytoplasmic tail to gain heterodimer selectivity with high femtomolar affinity towards Zn(II) to underpin the need to establish new routes for protein engineering, molecular biology, nanotechnology, etc. (Fig.…”