Proteins with calmodulin-like domains: structures and functional roles

Abstract

The appearance of modular proteins is a widespread phenomenon during the evolution of proteins. The combinatorial arrangement of different functional and/or structural domains within a single polypeptide chain yields a wide variety of activities and regulatory properties to the modular proteins. In this review, we will discuss proteins, that in addition to their catalytic, transport, structure, localization or adaptor functions, also have segments resembling the helix-loop-helix EF-hand motifs found in Ca²⁺-binding proteins, such as calmodulin (CaM). These segments are denoted CaM-like domains (CaM-LDs) and play a regulatory role, making these CaM-like proteins sensitive to Ca²⁺ transients within the cell, and hence are able to transduce the Ca²⁺ signal leading to specific cellular responses. Importantly, this arrangement allows to this group of proteins direct regulation independent of other Ca²⁺-sensitive sensor/transducer proteins, such as CaM. In addition, this review also covers CaM-binding proteins, in which their CaM-binding site (CBS), in the absence of CaM, is proposed to interact with other segments of the same protein denoted CaM-like binding site (CLBS). CLBS are important regulatory motifs, acting either by keeping these CaM-binding proteins inactive in the absence of CaM, enhancing the stability of protein complexes and/or facilitating their dimerization via CBS/CLBS interaction. The existence of proteins containing CaM-LDs or CLBSs substantially adds to the enormous versatility and complexity of Ca²⁺/CaM signaling.

Keywords: Calcineurin; Calpain; Epidermal growth factor receptor; Glycerol-3-phosphate dehydrogenase; NADPH oxidases; Na+/H+ exchanger; Plasma membrane Ca2+-ATPase; Protein kinases; α-Actinin.

Fig. 1

Domain organization of some calmodulin-like domain (CaM-LD)- and calmodulin-like binding site (CLBS)-containing proteins with enzymatic activity. The figure depicts the linear organization of the relevant domains of some enzymes with either a CaM-LD or CLBS. The domains shown are: catalytic site (red); CaM-LD or CLBS (green and EF-hands, boxes with bars, where applicable); autoinhibitory (AI)/junction (JD) domains (magenta); and calmodulin-binding site (CBS) (pink). CDPK CaM-like domain protein kinase, CaMK-II CaM-dependent protein kinase-II, MLCK myosin light-chain kinase, EGFR epidermal growth factor receptor, CaN A calcineurin A, CaN B calcineurin B, Calp 80 calpain 80 kDa subunit, Calp 30 calpain 30 kDa subunit, GAPDH glycerol-3-phosphate dehydrogenase, NOX5 NADPH oxidase 5, PMCA plasma membrane Ca²⁺-ATPase. The length of the proteins and different domains is not drawn to scale. The CBS and AI domains of the EGFR overlap [183]

Fig. 2

Structure and Ca²⁺-mediated activation of a calmodulin-like domain protein kinase. a The figure depicts the X-ray crystallographic structure of Ca²⁺-dependent protein kinase 1 (AtCDPK1) from Arabidopsis thaliana at 2 Å resolution (Q06850 CDPK1_ARATH based on template 4m97A, residue range 142–588) taken from UniProt SWISS-MODEL database. The crystallographic structure in the left panel is shown using a rainbow color code going from the N-terminal (blue) to the C-terminal (red). The Ca²⁺-binding sites in the four EF-hands are highlighted in the right panel in different colors indicating the amino acid residues involved. b The figure depicts a model for the Ca²⁺-dependent activation of a CaM-like domain protein kinase (CDPK). In the absence of Ca²⁺, a non-physiological condition, when the four EF-hands of the CDPK are free of Ca²⁺ (apo-CDPK), a sector of the junction domain (JD) blocks the catalytic site of the protein kinase (PK) rendering the enzyme inactive (top panel). In basal conditions, when the cytosolic Ca²⁺ concentration is very low (< 50–100 nM) the high-affinity Ca²⁺-binding sites located in the distal EF-hands 3 and 4 are occupied and both of them interact with the JD (marked in green), the catalytic site is partially released and the enzyme becomes partially active (central panel). The interrogation mark indicates a discrepancy in the literature as to whether binding of Ca²⁺ to EF-hands 3 and 4 is sufficient to at least partially release the auto-inhibition (see references [–31]). When the cytosolic Ca²⁺ concentration increases at saturating concentrations (> 0.5–1 µM), the two low-affinity Ca²⁺-binding sites located in EF-hands 1 and 2 are also occupied by Ca²⁺, and all EF-hands interact with the JD (marked in green) which is totally released from the catalytic site rendering the enzyme fully active (bottom panel). Ca²⁺ ions are represented by gray spheres. CaM-LD calmodulin-like domain. Partially based on references [31, 36]

Fig. 3

Structure and function of the Ca²⁺ binding sites of m-calpain, α-actinin, ATP/phosphate exchanger and calcineurin. a X-ray crystallographic structure of the rat m-calpain/calpain-2 dimer with Ca²⁺ bound in complex with the physiological inhibitor calpastatin at 2.4 Å resolution. The structure was obtained from PDB: ID 3BOW. The EF-hand with Ca²⁺-bound (gray spheres), the loop forming the acidic electrostatic switch (AES) and domains IIa, IIb, III and IV in the large catalytic subunit (pink), and domain VI in the small regulatory subunit (blue) are indicated [66]. The red arrow shows the cleft between the two catalytic sub-domains IIa and IIb [66, 67]. A segment (B-peptide, residues 595–629) of calpastatin (brown) blocking the catalytic site in domains IIa and IIb is also shown [68, 212]. b X-ray crystallographic structure of a human muscle α-actinin-2 (α-Act-2) dimer (residues 16–874) at 3.5 Å resolution is shown in pink and blue colors. The location of EF-hands 1 and 2 and the actin-binding domain (ABD) are indicated (left). Cryo-electron microscope derived structure of tandem calponin-homology domains (residues 1–109) of human skeletal muscle α-actinin-3 (α-Act-3) bound to a F-actin filament formed by G-actin subunits (residues 1–374) at 15 Å resolution. Each calponin-1 homology domain (CH) and actin subunit is represented in different colors (right). The structures were obtained from PDB ID: 4D1E and 3LUE, respectively [104, 213, 214]. c The CaM-like binding domain (CaM-BD), also denoted Ca²⁺ sensor, of human mitochondrial ATP/phosphate exchanger-1 (SCaMC-1) located at its N-terminus is shown in a rainbow color code (residues 22–176). The four EF-hands bound to Ca²⁺ (gray sphere) and diethylene glycol molecule are indicated. The structure was obtained from PDB ID 4N5X [121]. d The figure depicts the calcineurin A (CaN-A, pink)/calcineurin B (CaN-B, blue) heterodimer in which the four EF-hands of the regulatory CaN-B subunit are saturated with Ca²⁺ (cyan spheres), and the calmodulin-binding site (CBS) of CaN-A (residues 391–414, dashed box) is already separated from the CaN-B binding region (residues 348–370) considered to be a calmodulin-like binding site (CLBS). Binding of the Ca²⁺/calmodulin complex (Ca²⁺/CaM, rainbow color coded) to the CBS allows the C-terminal auto-inhibitory site (AIS) (residues 457–482) to be separated from the Zn/Fe-containing (brown spheres) catalytic site (residues 71–342) releasing the auto-inhibition and inducing full activation of the enzyme. The structures were obtained from PDB ID: 1AUI (human CaN-A/CaN-B heterodimer at 2.1 Å resolution) [215] and 1CLL (human CaM at 1.7 Å resolution) [216]. See text and Ref. [135] for more details on the activation mechanism

Fig. 4

Structure and CBS/CLBS location in CaMK-II and MLCK. The figure depicts two chains of the autoinhibited dodecameric human CaMK-II δ isoform showing the contiguous autoinhibitory domain (AID) and CBS in both chains (a); the X-ray crystallographic structure and the location of the calmodulin-binding domain (CBS) and calmodulin-like binding site (CLBS) of the β-subunit of calmodulin-dependent protein kinase-II (CaMK-II) from rat (b); and myosin light-chain kinase-2 (MLCK-2) from rabbit skeletal/cardiac muscle showing the CBS and CLBS (c). In panels b and c left the structures are shown using a rainbow color code going from the N-terminal (blue) to the C-terminal (red), and in panels b and c right highlighting in color the CBS and CLBS. In panel a two chains of CaMK-II are shown in different colors. Crystallographic 3D models for rat and human CaMK-II at 2.3 Å resolution (P08413 KCC2B_RAT based on template 2vn9A, residue range 10–309, and Q13557 KCC2D_HUMAN based on template 2vn9, residue range 11–309), and rabbit MLCK-2 at 2.2 Å resolution (P07313 MYLK2_RABIT based on template 2x0gA, residue range 290–596) were taken and modified from UniProt SWISS-MODEL database

Fig. 5

Structure and functional role of CBS/CLBS interaction in the EGFR. a The figure depicts the X-ray crystallographic structure of the cytosolic region of the human epidermal growth factor receptor (EGFR_cyt) at 3.2 Å resolution (P00533 EGFR_HUMAN based on template 3rcdA, residue range 702–1015 corresponding to residues 678–991 in the mature receptor lacking the 24 amino acids of the signal peptide) taken and modified from UniProt MODBASE database using a rainbow color code (left panel) going from the N-terminal (blue) to the C-terminal (red), and the location of the CaM-like domain (CLBS) highlights in color starting at Asp979 (right panel). b At resting basal conditions (left panel), the monomeric ligand-free epidermal growth factor receptor (EGFR) has the positively charged juxtamembrane calmodulin-binding domain (CBS) (blue segment) electrostatically bound to the negatively charged inner leaflet of the plasma membrane (minus symbols), which is rich in acidic phosphoinositides, maintaining the receptor in an auto-inhibited state [183]. Upon binding of the ligand epidermal growth factor (EGF) (central panel), the receptor initiates its dimerization and the Ca²⁺/CaM complex binds to the CBS helping to its detachment from the inner leaflet of the plasma membrane [183], and therefore contributing to the ligand-induced EGFR activation by trans-phosphorylation of C-terminal tyrosine residues (-Y-P). In this model, we propose that the EGFR dimer is active but in a quasi-stable conformation. Subsequently, the active EGFR dimer releases the Ca²⁺/CaM complex and adopts a more stable conformation (right panel) by the electrostatic interaction between of positively charged CBS (blue segment) of one EGFR monomer (labeled 1) with the negatively charged CaM-like domain (CLBS) (red segment) of the apposed EGFR monomer (labeled 2). For clarity, the N- and C-termini, transmembrane region (yellow segment), CBS, CLBS, and tyrosine kinase domain (TK) of each EGFR monomer are labeled with numbers 1 and 2 to document CBS/CLBS hetero-interaction of apposed monomers. Ca²⁺ ions are represented by gray spheres. ext extracellular medium, cyt cytosol. Adapted from Ref. [173]

Fig. 6

Structure and functional role of CBS/CLBS interaction in the plasma membrane Ca²⁺-ATPase. a The figure depicts the X-ray crystallographic structure of human plasma membrane Ca²⁺-ATPase isoform 1 (PMCA1) at 3 Å resolution (P20020 AT2B1_HUMAN based on template 2c9mA, residue range 52–1063) taken from UniProt SWISS-MODEL database. The crystallographic structure in the left panel is shown using a rainbow color code going from the N-terminal (blue) to the C-terminal (red), and in the right panel highlights in different colors the ten transmembrane segments. b At low cytosolic Ca²⁺ concentration the positively charged CaM-binding site (CBS) (blue segment), located at the C-terminal tail of the enzyme, is free of calmodulin (CaM) and interacts with two acidic regions representing a bi-partite calmodulin-like binding site (CLBS) (red segments), respectively located in the first and second intracellular bulky loops of the enzyme. The first loop goes between the second and third transmembrane region, and the second loop goes between the fourth and fifth transmembrane region (yellow segments). The CBS has two interaction sites separated by a 38 amino acids segment (not shown). The ten transmembrane regions are numbered, and the N- and C-termini of the enzyme indicated. The interaction of the CBS with the CLBS maintains the enzyme in an auto-inhibited state (left panel). When the cytosolic Ca²⁺ concentration increases the Ca²⁺/CaM complex is formed, binding with high affinity to the CBS and detaching itself from the CLBS, which renders the enzyme active (right panel). Ca²⁺ ions are represented by gray spheres. ext extracellular medium, cyt cytosol, PMCA plasma membrane Ca²⁺-ATPase. Adapted from Ref. [217]

Fig. 7

Structure and functional role of CBS/CLBS interaction in the Na⁺/H⁺-exchanger. a The figure depicts the X-ray crystallographic structure of rat Na⁺/H⁺-exchanger 1 (NHE1) at 3.5 Å resolution (P26431 SL9A1_RAT based on template 1zcda, residue range 109–510) taken from UniProt MODBASE database using a rainbow color code (left panel) going from the N-terminal (blue) to the C-terminal (red), and the location of eleven transmembrane and one intramembrane segment highlighted in different colors (right panel). b In resting conditions, the Na⁺/H⁺-exchanger 1 (NHE1) remains auto-inhibited (top panel) as the distal autoinhibitory/calmodulin-binding site (AI/CBS₂) has autoinhibitory function interacting with the proton modifier site (PMS) (dashed arrow), located in an intramembrane loop region. It has been hypothesized that CBS₁ (proximal CBS) interacts electrostatically with the distal-located negatively charged CaM-like site (CLBS) (marked with a question mark). When the cytosolic Ca²⁺ concentration increases the Ca²⁺/CaM complex is formed, binding to both the proximal and distal CBSs (CBS₁ and CBS₂, respectively) the autoinhibition is released by detachment of AI/CBS₂ from the regulatory PMS, rendering the NHE1 active (bottom panel). Ca²⁺ ions are represented by gray spheres. ext extracellular medium, cyt cytosol. The twelve transmembrane segments (yellow) are numbered, and the N- and C-termini of the exchanger are indicated. Adapted from references [–193]