Review 29 December 2020 , , and 1 Center for Diagnostics and Therapeutics, Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA 2 Chemistry Division, Georgia Gwinnett College, Lawrenceville, GA 30043, USA * Author to whom correspondence should be addressed.
Calmodulin (CaM) is an important intracellular protein that binds Ca 2+ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM’s ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca 2+ concentration through interactions with CaM.
Graphical Abstract
Calmodulin (CaM) is an intracellular Ca 2+-binding protein (CaBP) in eukaryotic systems, which functions as a second messenger that regulates myriad vital biological processes through interactions with more than 300 target proteins and peptides. The CaM protein, encoded by three different genes [1], is primarily expressed in eukaryotic organisms, and is one of most highly conserved protein sequences, with only histone proteins H4 and H3, actin B, and ubiquitin exhibiting greater evolutionary conservation. Genetic mutations to the CaM sequence can lead to various and potentially fatal pathologies, including ventricular tachycardia [2], congenital arrhythmia [3], and long QT syndrome [4]. These mutations can alter CaM’s affinity for binding Ca 2+ and its target proteins and peptides, thus interfering with their downstream activity.
Structurally, CaM is a predominantly helical protein (Figure 1A) that can be divided into N- and C-terminal domains. Each domain includes two paired Ca 2+-binding EF-hand motifs (Figure 1B). Each motif consists of a canonical helix-loop-helix (HLH) structure. The EF-hand motif, exhibiting pentagonal-bipyramidal geometry, includes a highly-conserved sequence of 12 amino acid residues, identified by relative positions 1–12. Six of these provide oxygen atoms as the preferred ligands for coordination of Ca 2+ ions [5] from side chains of residues in relative positions 1, 3, 5, and 12 [6], with oxygen from a carbonyl group in position 7. In addition to oxygen ligands from the amino acids, water molecules participate in forming the Ca 2+-stabilizing coordination complex [7]. The two domains are connected by an extended helix (Figure 1B) that is observed to be partially unwound and coiled in the Ca 2+-free state of the protein (Figure 1A) [8]. It has been experimentally verified that the extended helix has a propensity to be inherently disordered, increasing the overall flexibility of the protein, and allowing CaM to achieve different conformational states in its interactions with other peptides (Figure 1C–I) [9].
Figure 1. All CaM structures shown in blue, and CaMBPs are shown in red or tan. (A). Apo-CaM (PDB ID 1cfd) with central helix unwound/extended. (B). Holo-CaM (PDB ID 4bw8) with 4 Ca 2+ ions in EF-Hand sites 1–4. (C). Ca 2+/CaM/MARCKS (myristoylated alanine-rich C kinase substrate) complex (PDB ID 1iwq). The N-lobe of CaM is not involved in binding [10]. (D). Ca 2+/CaM/CaMKIIα complex (PDB ID 1cm1) exhibiting 1-5-10 binding mode with collapsed CaM [11]. (E). Ca 2+/CaM/RyR1 complex (PDB ID 2bcx) exhibiting unusual 1-17 binding mode with collapsed CaM [12]. (F). Ca 2+/CaM bound to peptide analog of CaM-binding region of chicken smooth muscle myosin light-chain kinase (PDB ID 1cdl) [13]. (G). Apo-CaM/Myosin V 2:1 complex (PDB ID 2ix7). Each CaM C- lobe is partially open to grip the first part of the IQ motif (IQxxxR), while the closed N-terminal lobes interact weakly with the second part of the motif (GxxxR) [14]. (H). Apo-CaM (blue) bound to zebrafish IQCG protein (red) (PDB ID 4lzx), exhibiting lower affinity than the Ca 2+-bound state [15]. Sidechain interactions with CaM include residues highlighted in bold from the IQCG sequence 400-410 (LQAWWRGTMIR). (I). Ca 2+/CaM/Glutamate decarboxylase chains B and C (PDB ID 1nwd) [16].
In the cell, CaM responds rapidly to increases in Ca 2+ concentrations, which normally ranges from approximately 10–100 nM, by binding up to four Ca 2+ ions in its paired EF-hand sites. The EF-hand pairs interact cooperatively [17], and it has been proposed that cooperativity between two coupled EF-hand binding sites of domains of CaM, causes global conformational changes in CaM that are conducive to binding target effector proteins to CaM [18]. The affinity range for each allosteric site has dissociation constant (K d) values between 10−7 and 10−11 M, indicating high affinity binding [19]. In general, the C-terminus motifs have a greater affinity for Ca 2+ than for the N-terminus motifs [20,21]. Affinity equilibriums at each binding site, in addition to being sensitive to allosterically-induced conformational changes, are also responsive to whether or not CaM has formed a binding complex with another protein. These changes are binding protein specific. Thus, there is a significant, cooperativity-mediated sensitivity to Ca 2+ that is both inherent to CaM’s native conformation and influenced considerably by intraspecific and interspecific factors.
The Ca 2+/CaM complex alters the conformation of CaM to interact with CaM-binding domains (CaMBDs) of target proteins and peptides [22]. It is through interactions with numerous CaM-binding proteins that CaM regulates diverse physiological processes that include memory formation [23], muscle contraction [24], cellular metabolism [25], and cytoskeletal rearrangements [26]. The proteins that CaM interacts with have been found in many different cellular locations and physiological environments. Myosin light-chain kinase [24], calcineurin [27], and CaM-dependent kinases I–IV [28] are cytosolic calmodulin effector proteins involved in motility, protein dephosphorylation, and protein phosphorylation processes, respectively. Recent rapid developments in structural, genomic, and analytical methods have revealed the important roles of CaM in regulating membrane proteins, including Na v 1.2/1.5 channels [20,21], IP 3 R channels [29], connexins [30], and GPCRs. These membrane embedded proteins participate in cellular depolarization, intracellular second-messenger signaling, and paracellular signaling, respectively.
In this review, we will first provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. We will then review prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. Historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction, will also be discussed.
The properties of CaM that facilitate its binding modes include the flexible central linker domain [31], methionine-rich linker domain, helix-helix movement, and side chain rearrangements [32]. The N-lobe of CaM participates in binding more effectively during complex formation associated with increases in local concentrations of Ca 2+, while the C-lobe participates more effectively at lower Ca 2+ concentrations. Thus chelation of Ca 2+ initiates Ca 2+-dependent conformational changes at these lobes through α-helix movement and rearrangements of side-chain contacts, which in turn affects interactions with CaM-binding proteins.
Recent work on calmodulin’s different binding complexes [33] has improved our current understanding of the mechanisms that result in the two CaM termini domains exhibiting different characteristics in binding complex modes. Despite this, a comprehensive understanding of exactly how CaM can interact with such a large number of proteins and peptides has yet to be established [34]. The use of spectral clustering system modeling, which evaluates the extent of binding (e.g., loose binding vs. compact binding) as a function of solvent exposure (hydrophobicity) and interhelical angles, led to three significant discoveries. First, shallow binding occurs more often in the Ca 2+-free (apo) forms of calmodulin, which results in more interactions between polar and charged residues on the calmodulin-binding protein interface. Secondly, the C-terminus of the calmodulin protein has very fixed conformations for protein binding that usually lead to more compact binding modes. Interestingly, the C-terminus of CaM typically exhibits higher Ca 2+ binding affinity than the N-terminus of calmodulin, which strongly suggests that the C-terminus holoproteins represent an intermediate mode for calmodulin. Similar to the apoprotein form of calmodulin, the third discovery demonstrated that the N-terminus of calmodulin is flexible and binds more loosely to calmodulin-binding proteins. It is likely that the low calcium and high calcium binding modes for calmodulin (apo and N-terminal, respectively) bind with lower affinity to more effectively adjust to changes in Ca 2+ concentration. Thus, CaM may bind targets either in its apo or holo forms, and because CaM is divided into two lobes (N- and C-terminal lobes), CaM may also functional in a partially saturated state, where not all of the four EF-Hand sites are occupied.
For CaM-binding proteins (CaMBPs), binding typically involves a disorder-to-order conformational change [22], and studies on the relationships between ion channels and CaM have revealed that structural disorder provides the flexibility required for the fine-tuned modulation needed to maintain intracellular homeostasis within the extracellular milieu [35].
Targets that bind to CaM interact through regions of positive charge, hydrophilic residues, and hydrophobicity in the helices [36]. The methionine-rich grooves in the linker domain allow for interaction with CaMBPs containing amphipathic α helices that attach to holo-CaM using a pair of hydrophobic anchors [12].
During interaction with targets, CaM may also be described as exhibiting either an extended or collapsed form. In the collapsed form, connection of two anchor residues in the CaM-binding motif may reduce the distance between the two domains from 50 Å to less than 10 Å [37]. Several variations of this anchoring pattern have been identified, where the binding domain of the CaMBP include at least two hydrophobic anchor residues. Examples of sequences containing these anchoring patterns (Table 1) include 1-10, 1-12, 1-14, 1-16 [38], 1-17 [12] and 1-10-14 [39]. An extensive review of known protein structures of CaMBPs in the Protein DataBank was previously reported by Tidow and Nissen [40]. A 1–5–10 binding mode for holo-CaM in the collapsed form, determined using peptide models, was observed with the α-subfamily connexins (Cx50p 141–166, Cx44p 132–153 and Cx43p 136–158) [41,42]. In its extended mode, Ca 2+-activated CaM interacts with myosin V Ca 2+ channels, Ca 2+ pumps, and SK channels (Small conductance Ca 2+-activated potassium channels), among others [41]. CaM also interacts with myosin light-chain kinase (MLCK), which binds with a 1-14 anchoring that allows for the N and C-terminals of calmodulin to wrap around the helix (Figure 1E) [43].
Table 1. CaM-Binding Motifs.
The IQ motif, as seen in myosin V Ca 2+ channels, can interact with CaM when it is Ca 2+ free [43] or only partially saturated with Ca 2+, and in some cases, in its holo state [21] (Figure 1G). Calmodulin-dependent protein kinase II (CaMKII) also possesses a more compact 1-5-10 hydrophobic residue anchor pattern [40] (Figure 1D). This form may have evolved because the autoinhibitory domains on these proteins require more compact binding to reactivate the phosphorylation sites on the protein. All of these different patterns (Table 1) are dependent upon the extent to which calcium binds to calmodulin, meaning that the activation, inhibition, or regulation of myriad calmodulin-interacting proteins are all Ca 2+ concentration-dependent processes. Thus, proteins with IQ motif binding patterns are likely activated in the absence of intracellular calcium (Figure 1H), whereas proteins with auto-inhibitory domains are more likely to be activated by high concentrations of calcium. Additional insight into distinctions between apo- and holo-CaM, and interactions with IQ motifs, was recently presented by O’Day et al. [34].
The activity of the skeletal muscle ryanodine receptor (RyR1) is inhibited as a result of binding of Ca 2+ to the C-lobe of the CaM protein (Figure 1E) [12], which facilitates binding of CaM to RyR1 at its N-terminus through residues P3614-3643 [45,46,47]. Thus, inhibition is regulated at lower Ca 2+ concentrations. CaM also interacts with cardiac Ry
