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 (K-d(av) = 2.5 x 10(-14) M) and its additional elevation in the C-terminally extended domain construct (K-d(av) = 3.1 x 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. (C) 2012 Elsevier Inc. All rights reserved.