Experimental Insight into the Structural and Functional Roles of the 'Black' and 'Gray' Clusters in Recoverin, a Calcium Binding Protein with Four EF-Hand Motifs.

A1 Originalartikel i en vetenskaplig tidskrift (referentgranskad)


Interna författare/redaktörer


Publikationens författare: Permyakov SE, Vologzhannikova AS, Nemashkalova EL, Kazakov AS, Denesyuk AI, Denessiouk K, Baksheeva VE, Zamyatnin AA, Zernii EY, Uversky VN, Permyakov EA.
Förläggare: Multidisciplinary Digital Publishing Institute (MDPI)
Publiceringsår: 2019
Tidskrift: Molecules
Volym: 24
Nummer: 13


Abstrakt

Recently, we have found that calcium binding proteins of the EF-hand
superfamily (i.e., a large family of proteins containing
helix-loop-helix calcium binding motif or EF-hand) contain two types of
conserved clusters called cluster I ('black' cluster) and cluster II
('grey' cluster), which provide a supporting scaffold for the Ca2+
binding loops and contribute to the hydrophobic core of the EF-hand
domains. Cluster I is more conservative and mostly incorporates aromatic
amino acids, whereas cluster II includes a mix of aromatic,
hydrophobic, and polar amino acids of different sizes. Recoverin is
EF-hand Ca2+-binding protein containing two 'black' clusters
comprised of F35, F83, Y86 (N-terminal domain) and F106, E169, F172
(C-terminal domain) as well as two 'gray' clusters comprised of F70,
Q46, F49 (N-terminal domain) and W156, K119, V122 (C-terminal domain).
To understand a role of these residues in structure and function of
human recoverin, we sequentially substituted them for alanine and
studied the resulting mutants by a set of biophysical methods. Under
metal-free conditions, the 'black' clusters mutants (except for F35A and
E169A) were characterized by an increase in the α-helical content,
whereas the 'gray' cluster mutants (except for K119A) exhibited the
opposite behavior. By contrast, in Ca2+-loaded mutants the
α-helical content was always elevated. In the absence of calcium, the
substitutions only slightly affected multimerization of recoverin
regardless of their localization (except for K119A). Meanwhile, in the
presence of calcium mutations in N-terminal domain of the protein
significantly suppressed this process, indicating that surface
properties of Ca2+-bound recoverin are highly affected by
N-terminal cluster residues. The substitutions in C-terminal clusters
generally reduced thermal stability of recoverin with F172A ('black'
cluster) as well as W156A and K119A ('gray' cluster) being the most
efficacious in this respect. In contrast, the mutations in the
N-terminal clusters caused less pronounced differently directed changes
in thermal stability of the protein. The substitutions of F172, W156,
and K119 in C-terminal domain of recoverin together with substitution of
Q46 in its N-terminal domain provoked significant but diverse changes
in free energy associated with Ca2+ binding to the protein:
the mutant K119A demonstrated significantly improved calcium binding,
whereas F172A and W156A showed decrease in the calcium affinity and Q46A
exhibited no ion coordination in one of the Ca2+-binding
sites. The most of the N-terminal clusters mutations suppressed membrane
binding of recoverin and its inhibitory activity towards rhodopsin
kinase (GRK1). Surprisingly, the mutant W156A aberrantly activated
rhodopsin phosphorylation regardless of the presence of calcium. Taken
together, these data confirm the scaffolding function of several
cluster-forming residues and point to their critical role in supporting
physiological activity of recoverin.


Senast uppdaterad 2020-17-01 vid 04:33