ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 8, pp. 1376-1395 © Pleiades Publishing, Ltd., 2026.
1376
REVIEW
EF-Hand Calcium-Binding Motif:
Novel Structural and Functional Insights
Konstantin A. Denessiouk
1
, Evgeny A. Permyakov
1
, Alexander I. Denesyuk
1
,
and Sergey E. Permyakov
1,a
*
1
Institute for Biological Instrumentation, Russian Academy of Sciences,
Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences,
142290 Pushchino, Russia
a
e-mail: permyakov.se@ya.ru
Received April 9, 2026
Revised July 7, 2026
Accepted August 10, 2026
Abstract— The EF-hand motif is one of the most widespread calcium-binding protein motifs in nature,
mediating calcium signaling across a variety of biological processes. While structural and functional prop-
erties of the individual proteins within the EF-hand superfamily are well studied, general principles gov-
erning organization and function of these proteins are only now being elucidated. This review examines
some of these emerging patterns, including recently discovered structural elements of the EF-hand mo-
tif (EF-hand zone, one-residue and three-residue units, local “Clamp” units, and “black” and “gray” clus-
ters). Additionally, we discuss the ability of certain EF-hand proteins to recognize a broad spectrum of
protein targets, as well as their zinc-binding properties. New structural data on the EF-hand domain al-
low proposing a structural-functional classification for the proteins in this family. Furthermore, tenden-
cy of some EF-hand proteins toward promiscuity and zinc binding significantly expands their functional
importance.
DOI: 10.1134/S0006297926601115
Keywords: three-dimensional structure, protein structure, motif, cluster, protein–ligand interactions, protein–
protein interactions, promiscuity, calcium, zinc, calcium-binding proteins, EF-hand, S100 proteins, calmodulin
* To whom correspondence should be addressed.
INTRODUCTION
Calcium ions are present in all living organ-
isms, fulfilling both structural and regulatory roles
[1-3]. Being a major component of hydroxyapatite in
bone and dental tissues, calcium is also essential for
maintaining active conformation of certain proteins,
such as in the β-propeller domains of integrin α-sub-
units and homologous bacterial domains [4]. Calcium
serves as a universal signaling messenger in cells,
regulating many critical aspects of cellular activity,
from fertilization to cell death at the end of their
life cycle [1-3, 5-7]. Calcium ions act as secondary
messengers released in response to the stimuli from
extracellular/intracellular stores, thereby serving as
signaling elements recognized by a wide range of
Ca
2+
-binding proteins in eukaryotes, prokaryotes, and
viruses. Calcium can also act as a primary messenger
by interacting with the receptors on the outer surface
of the plasma membrane [8].
Calcium ions regulate contraction of skeletal and
cardiac muscles: Ca
2+
binding to troponin C triggers
interaction between actin and myosin [9, 10]. In-
crease in the intracellular Ca
2+
concentration plays a
central role in signaling in the nervous system, trig-
gering neurotransmitter release [11]. Many processes
in the photoreceptor system are regulated by calci-
um ions, including functioning of ion channels, inac-
tivation of visual pigments, regulation of guanylate
cyclase activity, and others [12-14]. Ca
2+
ions control
gene transcription [15, 16] and many other biological
processes. Diverse activities of Ca
2+
are mediated by
the Ca
2+
-binding proteins, which perform the follow-
ing primary functions (see review [3]):
EF-HAND CALCIUM-BINDING MOTIF 1377
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 1. Three-dimensional structure of the Ca
2+
-bound EF-hand motif: single (a) and paired (b) motifs (PDB entry 1EXR).
Calcium ions are indicated by yellow spheres.
1. Ca
2+
-sensing function: converting changes in free
Ca
2+
levels into structural changes in the interact-
ing partners;
2. Ca
2+
buffering role: modulating local concentra-
tions of free Ca
2+
;
3. Transport of Ca
2+
ions.
Currently, several Ca
2+
-binding motifs in pro-
teins are known, with their profiles and amino acid
sequences available in the PROSITE database (https://
prosite.expasy.org) [17, 18]. One of the most com-
mon Ca
2+
-binding motifs is the DxDxDG loop [19, 20].
Among proteins with the DxDxDG loop, the EF-hand
family occupies an important place (Fig. 1a), con-
sisting of proteins with length of approximately 30
amino acids and featuring a helix-loop-helix struc-
ture [3]. The EF-hand motif was first discovered by
Prof. Kretsinger (R.H. Kretsinger) during the X-ray
structural analysis of parvalbumin [21]. He proposed
to model this structure as a right hand, where the
thumb and index finger represent helices, and the
bent middle finger represents the Ca
2+
-binding loop.
Since this model was based on the helices E and F
of parvalbumin, the motif was named the “EF-hand.”
In the paired EF-hand motif (Fig. 1b), two antiparallel
Ca
2+
-binding DxDxDG loops are surrounded by “enter-
ing” and “exiting” α-helices [22-24]. TheEF-hand motif
is found in more than 195 unique protein structures
(see the EF-hand superfamily – entry 3001983 in the
Structural Classification of Proteins (SCOP) database
[25, 26]) and is functional in all domains of life [27].
It allows proteins to sense changes in intracellular
free Ca
2+
concentrations from nanomolar to micromo-
lar levels and, accordingly, transmit calcium signals
to their binding partners, including enzymes, recep-
tors, ion channels and pumps, transcription factors,
and others [2, 3, 6]. Widespread presence and func-
tional diversity of the EF-hand proteins are associated
with their involvement in the development of several
socially significant diseases [28-40].
Decades of intensive research of various aspects
of functioning of the individual members of the EF-
hand family have provided general understanding of
their mechanisms of action at the cellular and organ-
ismal levels. This is particularly true for the calm-
odulin-like proteins, S100 proteins, and members of
the neuronal calcium sensor family. This knowledge
has allowed some of these proteins to be introduced
into clinical practice as diagnostic markers and has
initiated development of the targeted drugs aimed at
specific mechanisms of their action in the body [41].
However, over the past 10-15 years, new data have
emerged regarding the structural and functional fea-
tures of EF-hand proteins, allowing us to view this
protein family from a broader perspective. This re-
view is devoted to consideration and discussion of
some of these data.
STRUCTURAL ELEMENTS
OF THE EF-HAND MOTIF
Spatial structures of the EF-hand domains con-
tain a characteristic linear motif DxDxDG, or more
precisely, Dx[DN]xDG, which was identified in the
helix-loop-helix supersecondary structure among 18
different families of Ca
2+
-binding proteins [19, 20].
DENESSIOUK et al.1378
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 2. Ca
2+
-binding “EF-hand” zone, shown using calmodulin as an example. Key positions of the zone are labeled with
numbers I-VI. X, Y, Z, -X, -Y, and -Z denote the key Ca
2+
-binding amino acids in the Kretsinger coordinate system [21].
For simplicity, the side chain atoms of aspartate residues at positions I (Asp20) and II (Asp22) are omitted. Amino acids
(carbon atoms – gray, nitrogen – blue, oxygen – red), water molecules (red), and Ca
2+
ions (yellow) are depicted using a
ball-and-stick model. Contacts between atoms are indicated by dashed lines.
Based on this motif as a reference, we reconstructed a
mutually similar local environment around the bound
Ca
2+
ion for all the mentioned families of Ca
2+
-binding
proteins and presented it for calmodulin, whose spa-
tial coordinates were used as a representative struc-
ture of EF-hand domains [42]. This structural model
of Ca
2+
binding was named the “EF-hand zone,” which
includes the EF-hand motif [19, 20] and additional
amino acids connected by a network of interactions,
resulting in a circular closed Ca
2+
-binding site (Fig. 2).
In calmodulin, in addition to the Ca
2+
-binding
motif DKDGDG (positions I, II, and III in Fig. 2), the
next amino acid in position IV provides interaction
with Ca
2+
via the oxygen of the main chain (O/Thr26-
Ca
2+
contact). Note that positions I, II, III, IV, and V
in Fig. 2 correspond to positionsX, Y, Z, -Y, and-Z in
the Ca
2+
-binding loop coordinate system introduced
by Kretsinger [21]. Hereafter, we will use the coordi-
nate system based on the I-VI designations. The local
closed circular structure is stabilized by the known
Asx-motif (N/Thr26-OD1/Asp24 contact) [43]. Between
the N- and C-terminal regions of the “EF-hand” zone,
there is a glutamate residue (position V, Glu31 in
Fig. 2) that closes the loop around Ca
2+
. Number of
amino acids between the “EF-hand” zone and the
residue at position V can vary from 2 to 3, 6, 15,
56, and 62 for the “EF-hand”-like zones of various
proteins. This variability in the length of this region
significantly expands the range of tertiary structures
capable of including a Ca
2+
-binding “EF-hand”-like
zone while preserving its overall structure.
Interactions specific to the “EF-hand” zone of
calmodulin include two hydrogen bonds (N/Thr28-
OE1/Glu31 and N/Asp22-OE2/Glu31), three weak hy-
drogen bonds (O/Thr28-CB/Glu31, O/Phe19-CB/Glu31,
and OE1/Glu31-CA/Ile27), and coordination of the cal-
cium ion by OE1/Glu31-Ca
2+
. A water molecule also
coordinates Ca
2+
at positionVI. Thus, the Ca
2+
-binding
site of calmodulin consists of residues of the DxDxDG
motif and additional interactions at positions IV, V,
EF-HAND CALCIUM-BINDING MOTIF 1379
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 3. Types of local substructures for Ca
2+
recognition by “EF-hand” zones in proteins: a) one-residue unit type I (ORI);
b) three-residue unit type I (TRI); c) one-residue unit type II (ORII); and d) three-residue unit type II (TRII). Type I and
type II denote the method of Ca
2+
interaction with the local substructure. Two oxygen atoms directly coordinating Ca
2+
are designated as “position1” and “position2.” Thefunctional nitrogen atom of the main chain of each local substructure
is designated as “position 3.” In the type II substructures, Ca
2+
is bound between the oxygen atom of the main chain at
position1 and the nitrogen atom of the main chain at position3 via two oxygen atoms at positions 2 and 4. The bond be-
tween the atoms at positions2 and4 represents a rigid connection between two atoms of the same amino acid(n) or two
adjacent amino acids (n) and (n−1) or (n) and (n+1). Amino acid atoms and ligand atoms (carbon – gray, nitrogen – blue,
oxygen – red, and Ca
2+
– green) are depicted using a ball-and-stick model.
and VI (Fig. 2). In addition to the EF-hand superfam-
ily, whose representative is calmodulin, existence of
the “EF-hand” zone has been established in seven
other families of Ca
2+
-binding proteins: four of them
are similar to the Ca
2+
-binding site of calmodulin,
while in the remaining three families, position VI is
occupied not by water but by side chain atoms of
one of the residues of the same Ca
2+
-binding site[42].
The continuous Asp20-Thr28 fragment of the “EF-
hand” zone of calmodulin could be conditionally di-
vided into two parts: Asp20-Gly25 and Thr26-Thr28
(Fig. 2). The first part is formed by the Dx[DN]xDG
motif and contains three Ca
2+
-coordinating interac-
tions. The second part contains one additional in-
teraction: oxygen atom of the carbonyl group of the
main chain always interacts with Ca
2+
. It was estab-
lished in the early 1990s that most metal cations, and
in particular Ca
2+
, often interact with the oxygen at-
oms of the carbonyl groups of the main chain [44].
Additionally, two carbonyl groups of the main chain
could be connected by a metal ion as a bridge, which
is called a “niche” for metal binding [45].
DENESSIOUK et al.1380
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 4. Types of “Clamp” units for Ca
2+
binding: a) Clamp
n,(n−2)
, b) Clamp
n,(n+2)
, c) Clamp
n,(n−1)
, d) Clamp
n,(n+1)
, e) Clamp
n,n
.
The following structures were used to create the figures: aandb)parvalbumin (PDB 2PVB); c)fibroblast collagenase (PDB
1HFC); d) annexin III (PDB 1AXN); e) peroxidase (PDB 1GWU).
Analysis of the structure of metal cation-bind-
ing sites in proteins, conducted on 406 representa-
tive crystallographic structures from the PDB data-
base containing bound Ca
2+
atoms with resolution
of at least 1.5 Å for the proteins with amino acid
sequence identity not exceeding 30%, revealed four
minimal structural elements of the protein interac-
tions with Ca
2+
: the one-residue unit type I (ORI);
the three-residue unit type I (TRI); the one-residue
unit type II (ORII); and the three-residue unit type II
(TRII) (Fig. 3) [46]. In all types of the units, the main
atoms interacting with Ca
2+
are two oxygen atoms:
(i) the oxygen atom of the main chain at position 1
and (ii) the mediator atom at position 2. Difference
between the types I and II is that in the type I units,
there is one mediator atom, while in the typeII units,
there are two (Fig. 3). Depending on the nature of
the mediator atoms, there are four different variants
of the type I units and twelve variants of the type II
units [46]. Typically, a Ca
2+
-binding site in a protein
contains combination of the ORI/ORII and TRI/TRII
units, and the paired OR(I/II)+TR(I/II) unit forms a
tripeptide that coordinates Ca
2+
with three atoms of
the main chain: N
i
(nitrogen; donor) – O
i
(oxygen;
acceptor) – N
i+2
(nitrogen; donor), where i is the se-
quence number of the amino acid in the tripeptide.
Thus, the described ORI, TRI, ORII, and TRII units
serve as elementary “building blocks” for construct-
ing Ca
2+
-binding substructures in proteins.
Analysis of the amino acid composition of the
one-residue ORI and three-residue TRI units in dif-
ferent proteins showed that position 2 typically
EF-HAND CALCIUM-BINDING MOTIF 1381
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 5. Structural elements of the paired EF-hand motif. Conservative elements include: flanking entering and exiting α-he-
lices (helices I-IV), residues of the “black” and “gray” clusters (marked by black/gray circles), position of Ca
2+
-binding loops
DxDxDG, and central β-sheet. The structural coordinate system (X, Y, Z, -X, -Y, -Z, andX-4) corresponds to the key structural
positions of the EF-hand motif [21]. Supersecondary structure of the odd EF-hand of the helix-loop-helix type is marked
in gray.
contains oxygen of the side group of a polar amino
acid, which either precedes or follows, with one ami-
no acid in between, the amino acid that binds Ca
2+
via its carbonyl oxygen at position 1 (Fig. 3) [46].
From the group of 406 aforementioned structures, 25
structures containing various variants of ORI/II and
TRI/II units were selected. Weanalyzed the metal cat-
ion-binding sites in these proteins and additionally in
the Ca
2+
-binding proteins subtilisin Nat (PDB 3VYV)
and annexin V (PDB 2IE7), which have structural
organization of the Ca
2+
-binding sites different from
that of the homologous proteins– subtilisin Carlsberg
(PDB 1R0R) and annexin III (PDB 1AXN). As a result
of analysis of these structures, five local configura-
tions were identified that bind Ca
2+
with two bonds at
positions 1 and 2, named “Clamp” units: Clamp
n,(n−2)
,
Clamp
n,(n−1)
, Clamp
n,n
, Clamp
n,(n+1)
and Clamp
n,(n+2)
,
where n is the sequence number of the amino acid
that binds Ca
2+
via its carbonyl oxygen at posi-
tion 1 (Fig. 4) [47].
Simultaneous use of the variants of “Clamp”
units and the above-described one-residue (OR) and
three-residue (TR) units allows for a correct descrip-
tion of the structure of Ca
2+
-binding sites in both EF-
hand proteins and representative members of some
other superfamilies of Ca
2+
-binding proteins [47].
Structural units of this construction set necessarily
contain a basic amino acid whose main chain oxy-
gen binds Ca
2+
(position 1). However, in some cases,
there are Ca
2+
-binding sites that do not use the car-
bonyl oxygen of the main chain as a Ca
2+
acceptor.
Spatial structure of such Ca
2+
-binding sites cannot
be explained based on the structural units present-
ed above. Nevertheless, use of these structural units
on the principle of a LEGO construction set allows
describing spatial arrangement of most possible Ca
2+
acceptors in the proteins with various folds and func-
tions, which is important for developing approach-
es to the design of metal-binding sites in proteins.
An example of creating a classical EF-hand motif is
a chimera composed of individual structural units of
concanavalin A and cellulase CelT [42].
Structural models of the Ca
2+
-binding site de-
scribed above were based on the structure of one
“EF-hand” zone of one EF-hand motif. However, in
most known spatial structures, EF-hand motifs are
paired (Fig. 1b), and such EF-hand domains represent
a combination of two “EF-hand” zones [48] (Fig. 5).
The “EF-hand” zone that appears first along the
amino acid sequence is called the “odd” zone, and
the second is called the “even” zone [21]. N-termi-
nal and C-terminal flanking α-helices in each he-
lix-loop-helix substructure are called the “entering”
and “exiting” α-helices, respectively. Six residues of
the “EF-hand” zone that coordinate Ca
2+
are designat-
ed as X, Y, Z, and -X, -Y, -Z. In the study of eleven
representatives of EF-hand proteins, two symmetric
conservative clusters of interacting residues were
discovered that connect the entering and exiting
α-helices of the even and odd “EF-hand” zones [48].
These clusters connect the ends of the EF-hand do-
main and consist of interacting residues at posi-
tions X-4, -X+1, and -Z+1, named “Cluster I” or the
“black cluster” (black circles in Fig. 5) and “Cluster
II” or the “gray cluster” (gray circles in Fig. 5). The
“black” and “gray” clusters of EF-hand domains are
DENESSIOUK et al.1382
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
structurally and functionally nonequivalent. They
provide a framework for Ca
2+
-binding loops and con-
tribute to formation of the hydrophobic core of the
EF-hand domain.
The EF-hand domains could be classified based
on the behavior of the residues of the “black” and
“gray” clusters during Ca
2+
binding. Depending on
how local conformation of the “black” and “gray”
clusters (assessed by the contact surface area of ami-
no acid residues within the cluster) and global con-
formation of the EF-hand domain, reflected in the
relative arrangement of the clusters with respect to
each other, EF-hand domains could be divided into
the following groups [48]:
1. Open static (upon Ca
2+
binding, open conforma-
tion of the domain does not change, and local
structure of the clusters does not change; exam-
ples: S100P and parvalbumin);
2. Closed static (upon Ca
2+
binding, closed confor-
mation of the domain does not change, and local
structure does not change; example: S100A16);
3. Local dynamic (upon Ca
2+
binding, closed con-
formation of the domain changes to an open
one, and local structure also changes; examples:
S100B, S100A1, S100A4, S100A5, and S100A6);
4. Dynamic (upon Ca
2+
binding, closed conforma-
tion of the domain changes to an open one, but
local structure does not change; examples: calm-
odulin, troponin C);
5. Local static (upon Ca
2+
binding, open conforma-
tion of the domain does not change, but local
structure changes; example: S100A13).
It should be noted that the traditional classifica-
tion of the EF-hand proteins into calcium sensors and
calcium buffers is undergoing changes, as accumulat-
ed experimental data indicate that the “pure” calcium
buffers are a rare exception[49]. Forthis reason, the
proposed structural-functional classification of the
EF-hand domains allows for a more accurate quali-
tative characterization of the behavior of proteins in
this family, even at the level of individual paired EF-
hand domains.
STRUCTURAL AND FUNCTIONAL ANALYSIS
OFTHE “BLACK” AND “GRAY” CLUSTERS
IN EF-HAND PROTEINS
“Black” clusters of the EF-hand domain are high-
ly conserved and mainly contain aromatic amino ac-
ids, while the “gray” clusters are less conserved and
contain a mixture of aromatic, hydrophobic, and
polar amino acids [48]. In this regard, the “black”
cluster is likely important for maintaining structur-
al stability of the protein, while the “gray” cluster
could facilitate adaptation of the conformational and
dynamic properties of the EF-hand domain to ensure
a wide range of kinetic and equilibrium constants
for Ca
2+
binding, as well as recognition of the tar-
get. To test this hypothesis, the role of amino acid
residues of the “black” and “gray” clusters in main-
taining structural and functional status of the pro-
tein was experimentally studied in several members
of the EF-hand family using the alanine scanning
method [50-53].
Parvalbumin contains two paired EF-hand motifs
and belongs to the first group of EF-hand proteins –
open static [48]. Replacements of the residues in the
“black” and “gray” clusters of the rat β-parvalbumin
(Phe48, Ala100, Phe103 and Gly61, Leu64, Met87, re-
spectively) with alanine (except for the Ala100Val re-
placement) showed that replacements in the “black”
cluster, compared to those in the “gray” cluster, are
accompanied by the significantly more pronounced
changes in various properties of the protein, includ-
ing hydrodynamic radius of its apo-form, thermosta-
bility of the protein loaded with Ca
2+
/Mg
2+
, and af-
finity for Ca
2+
[51]. Similarly, analysis of the effect
of these mutations on the protein’s propensity for
intrinsic disorder showed that the local tendency for
intrinsic disorder and the overall level of predicted
disorder in the protein strongly depend on the re-
placements in the “black” cluster, whereas mutations
in the “gray” cluster have a less pronounced effect.
These results demonstrate that the amino acids of the
“black” cluster make a more significant contribution
to the maintenance of the structural and functional
properties of parvalbumin compared to the residues
of the “gray” cluster.
A similar study of another representative of the
open static group of EF-hand proteins containing one
paired EF-hand motif, the S100P protein[48], showed
that alanine replacements in its “black” and “gray”
clusters cause comparable changes in the individual
physicochemical properties of the protein [53]. How-
ever, experiments with the protein unfolding by heat
or denaturant showed that the replacements in the
“black” cluster cause significantly more pronounced
changes in the protein stability compared to those
caused by the replacements in the “gray” cluster.
The same effect has been observed regarding the
affinity of S100P for interleukin-11 [50]. Thus, as in
parvalbumin, the “black” cluster of S100P is more im-
portant for maintaining conformational stability and
functional activity than the “gray” cluster.
Alanine scanning of the residues of the “black”
and “gray” clusters of recoverin (an inhibitor of rho-
dopsin kinase in the visual system), a protein with
two paired EF-hand motifs, an N-terminal (inactive
EF1 motif paired with active EF2), and a C-terminal
(active EF3 motif paired with inactive EF4), showed
a more complex picture of the influence of mutations
EF-HAND CALCIUM-BINDING MOTIF 1383
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
on the protein’s properties [52]. This fact may be re-
lated to both the more complex structure of the pro-
tein and inactivity of its two EF-hands. Nevertheless,
in some cases, there are pronounced differences in
the consequences of replacements in the “black” and
“gray” clusters of the protein. For example, increase
in the content of α-helices in the Ca
2+
-loaded protein
in response to replacements in the N-terminal “black”
cluster exceeds the effect of mutations in the “gray”
cluster. At the same time, a decrease in thermostabil-
ity of the apo-protein with replacements in the C-ter-
minal “gray” cluster exceeds that of the apo-protein
with mutations in the “black” cluster. Similarly, re-
placements in the “gray” clusters more strongly affect
the protein’s affinity for Ca
2+
compared to the muta-
tions in the “black” clusters. Also, mutations in the
N-terminal “black” cluster more strongly reduce af-
finity of the Ca
2+
-loaded protein for membranes and
more significantly affect affinity of the apo-protein
for membranes. Unexpectedly, the W156A replace-
ment inverts functional activity of recoverin, causing
a sharp increase in the rhodopsin phosphorylation
regardless of the presence of Ca
2+
, which indicates
activation of the rhodopsin kinase by recoverin. Over-
all, the available data indicate critical role of the res-
idues of the “black” and “gray” clusters of recoverin
in maintaining its physiological activity.
MULTIPLE PROTEIN–PROTEIN INTERACTIONS
OFEF-HAND FAMILY MEMBERS
Diverse functional activities of the EF-hand pro-
teins are largely determined by their ability to inter-
act with various, and primarily, protein targets. One
of the most intriguing phenomena requiring expla-
nation at the molecular level is the ability of certain
EF-hand proteins to interact with many targets (the
phenomenon of promiscuity). A striking example of
this phenomenon is calmodulin (CaM), an extreme-
ly widely expressed and highly conserved classical
representative of the family of EF-hand proteins in
eukaryotes, capable of recognizing hundreds of pro-
tein targets, including many enzymes, receptors, ion
channels and pumps, as well as transcription factors,
allowing it to play a central role in the intracellu-
lar calcium regulation of many vital processes [54,
55]. Accordingly, disruptions in the calmodulin-de-
pendent processes are associated with numerous
congenital and acquired diseases, including heart
diseases, cancer, and neurodegenerative diseases
[29-31, 56].
Analysis of the structural patterns that enable
CaM to recognize multiple targets reveals impor-
tance of the following factors: (1) conformational
mobility of the central “linker” region of CaM con-
necting its N- and C-terminal paired EF-hand motifs
(Fig. 6a); (2) conformational flexibility of the regions
of target proteins that interact with CaM, in some
cases [57-62].
Another factor contributing to promiscuity in
the CaM–protein interactions is the structure of the
typical CaM interaction interface consisting of N- and
C-terminal clusters of methionine residues with 2-4
“anchor” large hydrophobic amino acid residues of
the target [65]. Conformational mobility of the latter
provides a wide conformational space for the target
recognition. Analysis of 35 representative structures
of calmodulin complexes with various targets showed
that the key residues of CaM in this process are me-
thionines of the N-terminal domain at positions 51,
71, and 72, as well as methionines of the C-domain at
positions 124, 144, and 145 (Fig. 7)[66]. Notably, these
methionines isolate the target from the hydrophobic
cores of both CaM domains containing the above-men-
tioned “black” and “gray” clusters. Probably for this
reason, target binding, like interaction with Ca
2+
, does
not affect these clusters, thereby classifying CaM as
a “dynamic” group of EF-hand proteins, considering
global structural rearrangements of CaM [48].
There are alternative ways for CaM to interact
with targets [67]. For example, in the absence of
“anchor” hydrophobic residues in the target, in the
case of proteins covalently modified by a lipid at the
N/C-terminus carrying multiple positively charged
residues, immersion of the lipid component of the
target into the hydrophobic cavities of the N/C-ter-
minal domains of the Ca
2+
-bound form of CaM has
been observed, which results in formation of elec-
trostatic contacts between the positively charged end
of the target and the negatively charged residues
of CaM [68]. In particular, the N-terminal myristoyl
group of CAP23/NAP22 binds to the surface composed
of the hydrophobic pockets of both domains of the
Ca
2+
-loaded CaM, while five lysine residues at the
N-terminus of the protein electrostatically interact
with the negatively charged surface of CaM. Similar-
ly, the C-terminal farnesyl group of KRAS4b immerses
into the hydrophobic pocket of the C-terminal domain
of Ca
2+
-bound CaM, while the C-terminus of KRAS4b,
carrying eight lysine residues, electrostatically inter-
acts with the acidic surface of CaM [68].
Unlike the “open” conformation of Ca
2+
-bound
CaM, in which hydrophobic residues of the protein
involved in the target recognition are exposed to the
solvent, the apo-form of the protein is in a “closed”
conformation, characterized by low accessibility of
its hydrophobic residues to the solvent [60]. Never-
theless, many CaM targets, such as those containing
the IQ motif, are capable of interacting with the apo-
CaM [69, 70]. Thus, CaM is functionally active inde-
pendently of Ca
2+
binding, and the latter, in essence,
DENESSIOUK et al.1384
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 6. Three-dimensional structures of Ca
2+
-loaded forms of CaM(a) and of the human S100P homodimer(b) (PDB entries
1UP5 and 1J55, respectively). Calcium ions are indicated by yellow spheres. Relationship between the number of interac-
tion partners of human small S100 proteins according to the IntAct database [63] and the promiscuity parameter of S100
proteins according to [64] (c).
expands conformational space of the CaM molecule
capable of recognizing targets. Since loading of calci-
um into the four “EF-hands” of CaM covers about two
orders of magnitude of free calcium concentration
in solution [71], CaM can adopt, depending on the
Ca
2+
level, a series of conformations differing in the
degree of loading with Ca
2+
ions, which additionally
expands the possibilities of the functional response
of the protein.
Combination of the above factors facilitates real-
ization of a wide spectrum of conformations of CaM
complexes with its binding partners, depending on
the target and the level of Ca
2+
, thereby enriching
the repertoire of functional consequences of these
EF-HAND CALCIUM-BINDING MOTIF 1385
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 7. Schematic presentation of the relative positions of “black” and “gray” clusters of the hydrophobic core of calm-
odulin and the layer of methionine residues of its N-terminal domain (a) and C-terminal domain (b) that recognize the
target protein in the complex of calmodulin with the peptide of myosin light chain kinase of smooth muscles (PDB 2O5G)
used as an example. Residues in parentheses correspond to the target. Dashed lines denote hydrophobic contacts on the
interaction surface.
interactions. Structural features of the CaM-binding
sites in the proteins identified to date allow predict-
ing ability of other proteins to recognize CaM [58],
thereby contributing to the establishment of the reg-
ulatory role of calcium/CaM in biological processes.
Another striking example of the pronounced
ability of EF-hand superfamily proteins to engage in
multiple interactions is one of the largest families of
this superfamily, the S100 protein family. In humans,
it contains 25 members, with 239 genes in total en-
coding the human EF-hand proteins (Swiss-Prot da-
tabase 2026_01 [72]). Total number of the targets of
small S100 proteins (21 in total) reaches 1899, with
the number of interaction partners for the individ-
ual proteins exceeding 200-300 (S100A2, S100P – see
Fig. 6c), according to the IntAct interaction data-
base [63]. For comparison, the number of targets for
the products of three CaM genes according to IntAct
is 91, 95, and 190, respectively.
S100 proteins are evolutionarily young regulato-
ry Ca
2+
-binding proteins of vertebrates, demonstrat-
ing tissue-specific/cell-specific expression and, similar
to CaM, participating in a wide variety of physiolog-
ical processes [36, 73-76]. S100 proteins contain a
canonical C-terminal EF-hand motif and N-terminal
EF-hand motif with the extended Ca
2+
-binding loop,
connected by a flexible “hinge” region [77] (Fig. 6b).
S100proteins form homo- or heterodimers and could
assemble into the higher-order multimers [78-80].
Ca
2+
binding causes conformational changes in S100
proteins, exposing hydrophobic residues important
for target recognition [77, 81]. Meanwhile, the apo-
form of some S100 proteins is also capable of protein–
protein interactions [82]. The S100 proteins perform
their functions both intracellularly (in the cytosol
and nucleus) and extracellularly, interacting with en-
zymes, transcription factors, ion channels, receptors,
cytokines, lipids, nucleic acids, and glycans, thereby
regulating many biological processes in a metal-de-
pendent manner [74, 76, 82-84]. With non-canonical
secretion or cell damage, individual S100 proteins ex-
hibit cytokine-like effects by recognizing a number of
receptors [74, 85-87]. Recent studies have also shown
that some S100 proteins directly interact with a va-
riety of interleukins, growth factors, colony-stimulat-
ing factors, and members of the tumor necrosis fac-
tor superfamily, in some cases affecting their cellular
activity [84, 85, 88-101]. S100 proteins are involved
in the development of a number of oncological, in-
flammatory, autoimmune, cardiovascular, pulmonary,
neurodegenerative, and metabolic diseases [32-40, 75,
102-104], with some of them being used as diagnostic
markers and investigated as therapeutic targets [33,
76, 105-108].
The tendency of S100 proteins toward multiple
interactions, explaining their multifunctionality, is
quantitatively characterized by the “promiscuity pa-
rameter.” This parameter represents a measure of
a protein’s ability to interact with a library of com-
pounds exposing combinations of two adjacent ami-
no acid residues for interaction [64]. This approach
allows dividing the proteins of S100 family into three
groups: proteins with high (S100A2, S100P, S100A5),
low (S100A7, S100G, S100Z), and medium promiscu-
ity tendencies. Similarly, S100 proteins were classi-
fied based on their peptide targets [109]. As seen in
Fig. 6c, promiscuity parameter of S100 proteins gen-
erally correlates with the number of their interaction
DENESSIOUK et al.1386
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
partners according to the IntAct database[63], allow-
ing the latter to be used to assess ability of the pro-
teins to engage in multiple interactions.
Unlike CaM, which has a high potential to adapt
its structure to a specific target due to conformational
mobility of the linker region connecting the N/C-ter-
minal EF-hand domains of CaM (Fig. 6a), compact
packing of the S100 protein dimers (Fig. 6b) and their
structural stability limit conformational possibilities
of S100 proteins during their interaction with targets.
Analysis of the structures of S100 protein complexes
with the peptides from their natural targets reveals
absence of a single binding site but presence of at
least two interaction sites within the S100 proteins
[81, 110]. Thefirst binding site in S100 proteins corre-
sponds to the hydrophobic surface between the heli-
cesIII andIV, exposed upon Ca
2+
binding. Thesecond
site consists of the helix I of one S100 monomer, as
well as helixIV and the “hinge” region of the second
monomer in the S100 dimer. Longer peptides could
use both of these sites for binding. The relatively
open interaction surfaces of S100 proteins with the
peptides and high variability in the amino acid se-
quences of the latter lead to different orientations
of the peptides relative to the S100 molecule. Pre-
sumably, each such interaction is based on a unique
combination of specific contacts of hydrophobic and
charged/polar side chains of the S100 protein and its
target. The recently resolved structure of the S100A4
protein complex with the transactivation domain of
the p53 protein generally corresponds to the data
obtained for the peptide targets: hydrophobic contact
surfaces of S100A4 are composed of the residues from
helices III and IV, as well as the “hinge” region[111].
In another complex of the S100 protein with a full-
length target, the RAGE receptor, the contact sites of
the S100A6 protein include residues from all helices
of the protein and the “hinge” [112]. The predicted
by molecular docking and confirmed by mutagenesis
binding site of the S100P protein for 20 4-helical cy-
tokines includes residues from the helicesI andIV of
the protein, as well as its “hinge” region [98]. A sim-
ilar binding site was found for the interaction of
the S100A6 protein with 30 4-helical cytokines [100].
Thus, S100 proteins demonstrate a wide repertoire of
structures of complexes with their peptide and full-
length targets.
An underappreciated factor contributing to mul-
tifunctionality of S100 proteins could be their pro-
pensity for intrinsic disorder, i.e., complete or partial
absence of a rigid tertiary structure, which provides
the protein with structural plasticity during interac-
tions with targets [113,114]. Inthe case of some S100
proteins, the propensity for disorder is not only pre-
dicted theoretically but also is manifested in the NMR
data, in the presence of unresolved regions in crystal
structures, as well as in the presence of post-trans-
lational modifications associated with the need to
ensure conformational freedom of the protein re-
gion undergoing modification [114]. Additionally, dis-
sociation of the protein dimer at its physiologically
significant concentrations should be accompanied
by a decrease in its structural stability, substantial
structural rearrangements leading to the changes
in its functional activity, as shown for the S100P
protein [114].
Examples of the structural plasticity of the S100
and CaM proteins in relation to recognition of the
protein targets demonstrate how diversity of func-
tional activities of the EF-hand family proteins is
achieved. Despite the described above structural pat-
terns of complex formation by CaM and S100 proteins
with their targets accurately predicting selectivity of
the interactions of these proteins with a particular
class of targets remains an unresolved problem, es-
pecially for the S100 proteins.
ZINC BINDING BY THE EF-HAND MOTIF
Another property of many EF-hand proteins
that determines their multifunctionality is their abil-
ity to bind zinc ions, along with Ca
2+
/Mg
2+
/Na
+
/K
+
ions [2, 3]. Zinc is a transition metal, so it is most
strongly bound by proteins through the sulfur atoms
of cysteine residues and nitrogen atoms of histidine
residues [115]. Interaction of zinc with its ligands, in
addition to electrostatic interactions, includes par-
tial overlap of electron clouds. There are three main
types of Zn
2+
-binding centers in proteins: structural,
catalytic, and co-catalytic. The ligands of zinc in these
centers are usually Cys, His, Asp, and Glu residues, as
well as of oxygen atoms of water. The geometry of
Zn
2+
-binding centers could vary from square to tetra-
hedral.
Zn
2+
-binding sites that do not coincide with the
calcium-binding sites are present in many EF-hand
proteins [116,117]. Such zinc-binding sites usually in-
clude sulfur atoms of cysteine residues and nitrogen
atoms of histidine residues. For example, each sub-
unit of the S100A3 dimer contains two Ca
2+
-binding
sites of the EF-hand type and one Zn
2+
-binding site
composed of three Cys and one His at the C-termi-
nus [118,119]. ItsCa
2+
-binding sites have low affinity
for calcium (dissociation constant in the millimolar
range), while a separate zinc-binding site binds Zn
2+
with dissociation constant in the micromolar range,
i.e., the protein is rather a Zn
2+
-binding protein than
a Ca
2+
-binding one. Similarly, a separate Zn
2+
-binding
site of the S100B dimer is formed by the residues His15
and His25 of one S100B subunit and residues His85
and Glu89 of the other subunit of the dimer [120].
EF-HAND CALCIUM-BINDING MOTIF 1387
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Fig. 8. Comparison of the structures of Ca
2+
and Zn
2+
binding sites in the EF-hand motifs1(a) and 3 (b) of the ALG-2 pro-
tein: PDB entries 2ZN9 (gray) and 2ZN8 (orange) for complexes with Ca
2+
and Zn
2+
, respectively.
While Ca
2+
binding by proteins generally stabilizes
their structure, Zn
2+
binding, on the contrary, desta-
bilizes their structure (see, for example, [121]).
Since zinc ions prefer Cys and His residues as li-
gands, and residues of both these amino acids are not
part of the Ca
2+
-binding sites, which prefer carboxyl
DENESSIOUK et al.1388
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
groups of Asp/Glu and carbonyl groups of the pro-
tein main chain, along with water molecules (oxygen
atoms are arranged approximately at the vertices of
a pentagonal bipyramid or tetrahedron), it is tradi-
tionally believed that Zn
2+
and Ca
2+
binding centers
are completely different. This view is supported by
the fact that ionic radii of calcium and zinc differ
significantly (0.99 Å and 0.74 Å, respectively), which
suggests substantial differences in the structure of
the coordination sphere of both metals. However,
some studies of the EF-hand proteins refute this point
of view. For example, Zn
2+
binding by the EF-hand
motifs of S100A1 and neuronal calcium sensor 1 has
been shown [122, 123]. Moreover, in some cases, the
X-ray diffraction method has established the structure
of the Zn
2+
-bound EF-hand motifs. For example, in
the study of ALG-2 protein, it was found that coordi-
nation of Zn
2+
in the EF1 and EF3 sites is similar to
coordination of Ca
2+
, except that the water molecule
in the coordination position -X of the motif EF1 is
absent in the case of Zn
2+
(Fig. 8) [124]. Additionally,
structures of the Ca
2+
/Zn
2+
-bound forms of the motif
EF3 differ in the position of residue Phe122. Thus,
replacement of a calcium ion with zinc in the EF-
hand motif could be accompanied by structural rear-
rangements capable of translating into a functional
response.
Similarly, the EF-hand motifs of the actin-binding
proteins EFhd1 and EFhd2 bind Zn
2+
with seven oxy-
gen atoms in the geometry of a distorted pentagonal
bipyramid in one binding site and in normal geome-
try of a pentagonal bipyramid in another site [125].
The paired EF-hand motifs of the N-terminal domain
of calmodulin also bind one Zn
2+
each [126]. One
of the Zn
2+
is tetrahedrally coordinated by the side
groups of residues Asp22 and Asp24 (which coordi-
nate calcium in the Ca
2+
-saturated protein), as well
as two molecules of the buffer. The second zinc ion
is also tetrahedrally coordinated by the side groups
of residues Glu67 and Asp64 and two groups of Glu7
and Glu11 of the neighboring molecule. Thus, four
out of five residues in the second binding loop used
for Ca
2+
binding do not participate in the interaction
with Zn
2+
. The overall structure of the Zn
2+
-bound
form of the protein differs significantly from the
structure of the Ca
2+
-saturated protein and is closer
to the structure of its apo-form.
Notably, affinity of Zn
2+
for the EF-hand motif
could significantly exceed that for Ca
2+
, opening the
possibility of competition between the two metals for
the same binding sites. For example, dissociation con-
stant (K
d
) of the S100A1 protein complex with Zn
2+
reaches 4 nM [122], making it possible to partially
fill the “EF-hands” of the protein with zinc, since
intracellular concentrations of labile zinc can reach
nanomolar levels [127]. Meanwhile, the K
d
for calci-
um ions of the S100A1 protein is only 0.2 µM [122],
which indicates partial loading of the protein with
calcium at the intracellular free calcium levels in the
resting state of 0.05-0.2 µM [128].
In summary, coordination of zinc ions by the EF-
hand motif could vary from tetrahedral to pentago-
nal bipyramidal, with the structural consequences of
zinc binding ranging from those close to rearrange-
ments during calcium binding to barely noticeable
rearrangements. This opens up the possibilities for
realization of the zinc-specific conformations of the
protein with functional activity different from that
of the Ca
2+
-bound or apo-form of the protein. Ad-
ditionally, competition between Ca
2+
and Zn
2+
ions
for the EF-hand motifs means that they represent a
point of intersection of calcium and zinc signaling
in the cell.
CONCLUSION
New structural patterns of the EF-hand motif
we have considered largely could be applied to the
structure of other superfamilies of Ca
2+
-binding pro-
teins, as well as to the binding of other metals, in-
cluding magnesium, potassium, sodium, cesium, etc.
Thediscovered structural patterns require further ex-
perimental study with various metal-binding proteins
to establish fundamental features of the structure of
metal-binding sites in proteins and role of their indi-
vidual structural elements in realization of the func-
tions of these proteins. This knowledge is also neces-
sary for designing proteins with specific properties
required to solve various applied problems.
As shown by the examples of calmodulin and
S100 proteins, such fundamental property of some
EF-hand proteins as promiscuity is determined by
employing a number of structural strategies. In ad-
dition to the protein–protein interactions considered
here, EF-hand proteins are also capable of interacting
with other classes of compounds, including lipids, nu-
cleic acids, and glycans. Insome cases, for realization
of such interactions, conformational flexibility of the
interaction partners is apparently important, which
occurs due to the presence of local intrinsically dis-
ordered regions in the binding partners. Another
phenomenon determining multifunctionality of the
EF-hand proteins is conformational rearrangement of
the protein in response to the binding of both calci-
um ions and zinc ions. Mixed Ca
2+
/Zn
2+
-bound forms
of the protein allow realization of many functionally
non-equivalent states of the protein, significantly ex-
panding functional significance of the EF-hand pro-
teins. Interrelationship of the calcium and zinc sig-
naling, at the center of which are the proteins of this
family, requires further experimental studies.
EF-HAND CALCIUM-BINDING MOTIF 1389
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 8 2026
Abbreviations
CaM calmodulin
Clamp clamp unit
EF-hand EF-hand motif (helix-loop-helix
motif)
OR one-residue unit
TR three-residue unit
Contributions
K. A. Denessiouk and S. E. Permyakov – concept;
K. A. Denessiouk, E. A. Permyakov, A. I. Denesyuk,
and S. E. Permyakov – writing the text.
Funding
This work was financially supported by the Russian
Science Foundation (grant no. 25-14-00223, S. E. Per-
myakov).
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest
The authors of this work declare that they have no
conflicts of interest.
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