Analyzing the structural and functional roles of residues from the 'black' and 'gray' clusters of human S100P protein. (June 2019)

• Record Type: Journal Article
• Authors: Permyakova, Maria E.; Permyakov, Sergei E.; Kazakov, Alexei S.; Denesyuk, Alexander I.; Denessiouk, Konstantin; Uversky, Vladimir N.; Permyakov, Eugene A.
• Is Part Of: Cell calcium. Volume 80(2019)
• Page Start: 46
• Page End: 55
• Publication Date: 2019-06
• Subjects: CD circular dichroism spectroscopy -- DSC differential scanning calorimetry -- DTT DL-dithiothreitol -- EDTA ethylenediaminetetraacetic acid -- EGTA ethylene glycol-bis2-aminoethylether-N, N, N', N'-tetraacetic acid -- ESI-MS electrospray ionization mass spectrometry -- HEPES N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) -- rWT S100P recombinant wild-type S100P -- PMSF phenylmethanesulfonyl fluoride -- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis -- Tris tris(hydroxymethyl) amino methane; S100P -- Clusters -- Structure -- Function -- Order -- Disorder; Calcium -- Metabolism -- Periodicals; Vertebrates -- Physiology -- Periodicals; Calcium -- Physiological effect -- Periodicals; Cell physiology -- Periodicals; Calcium in the body -- Periodicals; 572.516
• DOI: 10.1016/j.ceca.2019.03.008
• Languages: English
• ISSNs: 0143-4160

Abstract

Graphical abstract: Highlights: S100P cluster I is more important for protein conformational stability than the cluster II. F15A substitution in WT S100P did not cause the S100P dimer dissociation, indicating that F15 is not crucial for dimer stability. Analysis of heat- or GuHCl-induced unfolding of cluster mutants showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Abstract: Two highly conserved structural motifs observed in members of the EF-hand family of calcium binding proteins. The motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif represents a cluster of three amino acids called cluster I ('black' cluster) and cluster II ('grey' cluster). Cluster I is more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II is noticeably less conserved and includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. In the human calcium binding S100 P protein, these 'black' and 'gray' clusters include residues F15, F71, and F74 and L33, L58, and K30, respectively. To evaluate the effects of these clusters on structure and functionality of human S100 P, we have performed Ala scanning. The resulting mutants were studied by a multiparametric approach that included circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probes. Spectrofluorimetric Ca2+-titration of wild type S100 P showed that S100 P dimer has 1–2 strong calcium binding sites (K1 = 4 × 106 M −1 ) and two cooperative low affinity (K2 = 4 × 104 M −1 ) binding sites. Similarly, the S100 P mutants possess two types of calcium binding sites. This analysis revealed that the alanine substitutions in the clusters I and II caused comparable changes in the S100 P functional properties. However, analysis of heat- or GuHCl-induced unfolding of these proteins showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Opposite to literature data, the F15 A substitution did not cause the S100 P dimer dissociation, indicating that F15 is not crucial for dimer stability. Overall, similar to parvalbumins, the S100 P cluster I is more important for protein conformational stability than the cluster II.