Analyzing the structural and functional roles of residues from the 'black' and 'gray' clusters of human S100P protein

A1 Journal article (refereed)


Internal Authors/Editors


Publication Details

List of Authors: Permyakova ME, Permyakov SE, Kazakov AS, Denesyuk AI, Denessiouk K, Uversky VN, Permyakov EA
Publisher: Elsevier
Publication year: 2019
Journal: Cell Calcium
Volume number: 80
Start page: 46
End page: 55


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.


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