Femtomolar Zn2+ affinity of LIM domain of PDLIM1 protein uncovers crucial contribution of protein–protein interactions to protein stability
An individual LIM domain has approximately 55 amino acids with 8 highly conserved residues responsible for binding of two Zn2+ into two distinct zinc finger motifs. We examined LIM domain stability of PDLIM1 protein (known also as Elfin protein), its C-terminally extended constructs as well as separate zinc fingers, and several full domain mutants in terms of Zn2+ affinity and domain stability. Thermal denaturation, mass spectrometry, limited proteolysis, protein oxidation and circular dichroism techniques were used to determine a set of thermodynamic stability parameters. The results demonstrate unambiguously very high (femtomolar) affinity of both Zn2+ to the conserved LIM domain (Kdav = 2.5 × 10− 14 M) and its additional elevation in the C-terminally extended domain construct (Kdav = 3.1 × 10− 15 M). We demonstrate in the example of PDLIM1 using a set of LIM protein constructs and its zinc finger peptides that stability of the entire zinc-containing domain is not only defined by the Zn2+ coordination environment but significantly depends on the set of protein–protein interactions with the C-terminus of the protein. We discuss structural similarities of LIM domains and suggest the prolongation of the conserved LIM sequence to its C-terminal helix that has a significant impact on domain stability. We also discuss the functionality of LIM domains in terms of different physiological zinc and redox buffering capacity. LIM domain of PDLIM1 protein and its C-terminally extended constructs were examined in terms of Zn2+ affinity and protein stability. We showed that domain stability significantly depends on the set of protein–protein interactions. We discuss functionality of LIM domains in terms of different physiological zinc and redox buffering capacity. âº LIM domains have highly conserve residues responsible for binding of two Zn2+. âº LIM domain of PDLIM1 protein demonstrates femtomolar Zn2+ affinity. âº Zn2+ affinity is elevated by C-terminal tail of PDLIM1 protein. âº Extended LIM domain is fully saturated under wide range of cellular zinc and redox capacity. âº LIM domain is highly resistant to oxidation.