title: Femtomolar Zn2+ affinity of LIM domain of PDLIM1 protein uncovers crucial contribution of protein-protein interactions to protein stability.
abstract: An individual LIM domain has approximately 55 amino acids with 8 highly conserved residues responsible for binding of two Zn(2+) 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 Zn(2+) 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 Zn(2+) to the conserved LIM domain (K(d)(av)=2.5×10(-14) M) and its additional elevation in the C-terminally extended domain construct (K(d)(av)=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 Zn(2+) 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.
authors.affiliation: Department of Protein Engineering, Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Sikorska
authors.forename: Artur
authors.initials: A
authors.lastname: Krężel
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
mesh_terms: D006801:Humans; D060588:LIM Domain Proteins / Q000737:chemistry* / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014157:Transcription Factors; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: C484572:LDB2 protein, human; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D014157:Transcription Factors; D015032:Zinc