Gossypium hirsutum calmodulin-like protein (CML 11) interaction with geminivirus encoded protein using bioinformatics and molecular techniques.

Plant viruses are the most abundant destructive agents that exist in every ecosystem, causing severe diseases in multiple crops worldwide. Currently, a major gap is present in computational biology determining plant viruses interaction with its host. We lay out a strategy to extract virus-host protein interactions using various protein binding and interface methods for Geminiviridae, a second largest virus family. Using this approach, transcriptional activator protein (TrAP/C2) encoded by Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan virus (CLCuMV) showed strong binding affinity with calmodulin-like (CML) protein of Gossypium hirsutum (Gh-CML11). Higher negative value for the change in Gibbs free energy between TrAP and Gh-CML11 indicated strong binding affinity. Consensus from gene ontology database and in-silico nuclear localization signal (NLS) tools identified subcellular localization of TrAP in the nucleus associated with Gh-CML11 for virus infection. Data based on interaction prediction and docking methods present evidences that full length and truncated C2 strongly binds with Gh-CML11. This computational data was further validated with molecular results collected from yeast two-hybrid, bimolecular fluorescence complementation system and pull down assay. In this work, we also show the outcomes of full length and truncated TrAP on plant machinery. This is a first extensive report to delineate a role of CML protein from cotton with begomoviruses encoded transcription activator protein.

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

Investigating the Impact of Electrostatic Interactions on Calmodulin Binding and Ca2+-Dependent Activation of the Calcium-Gated Potassium SK4 Channel.

Ca2+ binding to the ubiquitous Ca2+ sensing protein calmodulin (CaM) activates the intermediate conductance Ca2+-activated SK4 channel. Potential hydrophilic pockets for CaM binding have been identified at the intracellular HA and HB helices in the C-terminal of SK4 from the three published cryo-EM structures of SK4. Single charge reversal substitutions at either site, significantly weakened the pull-down of SK4 by CaM wild-type (CaM), and decreased the TRAM-34 sensitive outward K+ current densities in native HEK293T cells when compared with SK4 WT measured under the same conditions. Only the doubly substituted SK4 R352D/R355D (HB helix) obliterated the CaM-mediated pull-down and thwarted outward K+ currents. However, overexpression of CaM E84K/E87K, which had been predicted to face the arginine doublet, restored the CaM-mediated pull-down of SK4 R352D/R355D and normalized its whole-cell current density. Virtual analysis of the putative salt bridges supports a unique role for the positively charged arginine doublet at the HB helix into anchoring the interaction with the negatively charged CaM glutamate 84 and 87 CaM. Our findings underscore the unique contribution of electrostatic interactions in carrying CaM binding onto SK4 and support the role of the C-terminal HB helix to the Ca2+-dependent gating process.

Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation.

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

Investigation of Structure-Stabilizing Elements in Proteins by Ion Mobility Mass Spectrometry and Collision-Induced Unfolding.

A recently developed proteolytic reactor, designed for protein structural investigation, was coupled to ion mobility mass spectrometry to monitor collisional cross section (CCS) evolution of model proteins undergoing trypsin-mediated mono enzymatic digestion. As peptides are released during digestion, the CCS of the remaining protein structure may deviate from the classical 2/3 power of the CCS-mass relationship for spherical structures. The classical relationship between CCS and mass (CCS = A × M2/3) for spherical structures, assuming a globular shape in the gas phase, may deviate as stabilizing elements are lost during digestion. In addition, collision-induced unfolding (CIU) experiments on partially digested proteins provided insights into the CCS resilience in the gas phase to ion activation, potentially due to the presence of stabilizing elements. The study initially investigated a model peptide ModBea (3 kDa), assessing the impact of disulfide bridges on CCS resilience in both reduced and oxidized forms. Subsequently, ß-lactoglobulin (2 disulfide bridges), calmodulin (Ca2+ coordination cation), and cytochrome c (heme) were selected to investigate the influence of common structuring elements on CCS resilience. CIU experiments probed the unfolding process, evaluating the effect of losing specific peptides on the energy landscapes of partially digested proteins. Comparisons of the TWCCSN2→He to trend curves describing the CCS/mass relationship revealed that proteins with structure-stabilizing elements consistently exhibit TWCCSN2→He and greater resilience toward CIU compared to proteins lacking these elements. The integration of online digestion, ion mobility, and CIU provides a valuable tool for identifying structuring elements in biopolymers in the gas phase.

Disruption of Ca2+/calmodulin:KSR1 interaction lowers ERK activation.

KSR1, a key scaffold protein for the MAPK pathway, facilitates ERK activation upon growth factor stimulation. We recently demonstrated that KSR1 binds the Ca2+-binding protein calmodulin (CaM), thereby providing an intersection between KSR1-mediated and Ca2+ signaling. In this study, we set out to generate a KSR1 point mutant with reduced Ca2+/CaM binding in order to unravel the functional implications of their interaction. To do so, we solved the structural determinants of complex formation. Using purified fragments of KSR1, we showed that Ca2+/CaM binds to the CA3 domain of KSR1. We then used in silico molecular modeling to predict contact residues for binding. This approach identified two possible modes of interaction: (1) binding of extended Ca2+/CaM to a globular conformation of KSR1-CA3 via electrostatic interactions or (2) binding of collapsed Ca2+/CaM to α-helical KSR1-CA3 via hydrophobic interactions. Experimentally, site-directed mutagenesis of the predicted contact residues for the two binding models favored that where collapsed Ca2+/CaM binds to the α-helical conformation of KSR1-CA3. Importantly, replacing KSR1-Phe355 with Asp reduces Ca2+/CaM binding by 76%. The KSR1-F355D mutation also significantly impairs the ability of EGF to activate ERK, which reveals that Ca2+/CaM binding promotes KSR1-mediated MAPK signaling. This work, by uncovering structural insight into the binding of KSR1 to Ca2+/CaM, identifies a KSR1 single-point mutant as a bioreagent to selectively study the crosstalk between Ca2+ and KSR1-mediated signaling.

Energetic and structural insights behind calcium induced conformational transition in calmodulin.

Calmodulin (CaM) is a key signaling protein that triggers several cellular and physiological processes inside the cell. Upon binding with calcium ion, CaM undergoes large scale conformational transition from a closed state to an open state that facilitates its interaction with various target protein and regulates their activity. This work explores the origin of the energetic and structural variation of the wild type and mutated CaM and explores the molecular origin for the structural differences between them. We first calculated the sequential calcium binding energy to CaM using the PDLD/S-LRA/ß approach. This study  shows a very good correlation with experimental calcium binding energies. Next we calculated the calcium binding energies to the wild type CaM and several mutated CaM systems which were reported experimentally. On the structural aspect, it has been reported experimentally that certain mutation (Q41L-K75I) in calcium bound CaM leads to complete conformational transition from an open to a closed state. By using equilibrium molecular dynamics simulation, free energy calculation and contact frequency map analysis, we have shown that the formation of a cluster of long-range hydrophobic contacts, initiated by the Q41L-K75I CaM variant is the driving force behind its closing motion. This study unravels the energetics and structural aspects behind calcium ion induced conformational changes in wild type CaM and its variant.

Revealing eEF-2 kinase: recent structural insights into function.

The α-kinase eukaryotic elongation factor 2 kinase (eEF-2K) regulates translational elongation by phosphorylating its ribosome-associated substrate, the GTPase eEF-2. eEF-2K is activated by calmodulin (CaM) through a distinctive mechanism unlike that in other CaM-dependent kinases (CAMK). We describe recent structural insights into this unique activation process and examine the effects of specific regulatory signals on this mechanism. We also highlight key unanswered questions to guide future structure-function studies. These include structural mechanisms which enable eEF-2K to interact with upstream/downstream partners and facilitate its integration of diverse inputs, including Ca2+ transients, phosphorylation mediated by energy/nutrient-sensing pathways, pH changes, and metabolites. Answering these questions is key to establishing how eEF-2K harmonizes translation with cellular requirements within the boundaries of its molecular landscape.

Controlling the interaction between CaMKII and Calmodulin with a photocrosslinking unnatural amino acid.

Using unnatural amino acid mutagenesis, we made a mutant of CaMKII that forms a covalent linkage to Calmodulin upon illumination by UV light. Like wild-type CaMKII, the L308BzF mutant stoichiometrically binds to Calmodulin, in a calcium-dependent manner. Using this construct, we demonstrate that Calmodulin binding to CaMKII, even under these stochiometric conditions, does not perturb the CaMKII oligomeric state. Furthermore, we were able to achieve activation of CaMKII L308BzF by UV-induced binding of Calmodulin, which, once established, is further insensitive to calcium depletion. In addition to the canonical auto-inhibitory role of the regulatory segment, inter-subunit crosslinking in the absence of CaM indicates that kinase domains and regulatory segments are substantially mobile in basal conditions. Characterization of CaMKIIL308BzF in vitro, and its expression in mammalian cells, suggests it could be a promising candidate for control of CaMKII activity in mammalian cells with light.

Calmodulin-Targeting Molecules from Ageratina grandifolia.

Four new natural chemical entities, including 2-hydroxy-α-truxillic acid (2), (3R,4S)-2,2-dimethyl-3-hydroxy-4-(1-angeloyloxy)-6-acetyl-7-methoxychromane (3), N-tricosanoyltyramine (4), and grandifolamide (5), were isolated along with 11 known compounds (1, 6-15) from the aerial parts of Ageratina grandifolia. The chemical structures were elucidated using chemical derivatization and HR-MS, NMR, and DFT-calculated chemical shifts, combined with DP4+ statistical analysis. It was found that 2 decomposed into its biogenetic precursor, o-coumaric acid, upon standing at room temperature for a few weeks. 3,5-Diprenyl-4-hydroxyacetophenone (8), O-methylencecalinol (10), encecalin (11), and encecalinol (12) bound to calmodulin (CaM) with higher affinity than chlorpromazine, a well-known CaM inhibitor. Molecular dynamics studies revealed that the complexes of these compounds with CaM remained stable during the simulation. Altogether these results revealed the therapeutic and research tool potential of compounds 8, 10, 11, and 12.

Structure of the complex between calmodulin and a functional construct of eukaryotic elongation factor 2 kinase bound to an ATP-competitive inhibitor.

The calmodulin-activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), serves as a master regulator of translational elongation by specifically phosphorylating and reducing the ribosome affinity of the guanosine triphosphatase, eukaryotic elongation factor 2 (eEF-2). Given its critical role in a fundamental cellular process, dysregulation of eEF-2K has been implicated in several human diseases, including those of the cardiovascular system, chronic neuropathies, and many cancers, making it a critical pharmacological target. In the absence of high-resolution structural information, high-throughput screening efforts have yielded small-molecule candidates that show promise as eEF-2K antagonists. Principal among these is the ATP-competitive pyrido-pyrimidinedione inhibitor, A-484954, which shows high specificity toward eEF-2K relative to a panel of "typical" protein kinases. A-484954 has been shown to have some degree of efficacy in animal models of several disease states. It has also been widely deployed as a reagent in eEF-2K-specific biochemical and cell-biological studies. However, given the absence of structural information, the precise mechanism of the A-484954-mediated inhibition of eEF-2K has remained obscure. Leveraging our identification of the calmodulin-activatable catalytic core of eEF-2K, and our recent determination of its long-elusive structure, here we present the structural basis for its specific inhibition by A-484954. This structure, which represents the first for an inhibitor-bound catalytic domain of a member of the α-kinase family, enables rationalization of the existing structure-activity relationship data for A-484954 variants and lays the groundwork for further optimization of this scaffold to attain enhanced specificity/potency against eEF-2K.

Growth or survival: What is the role of calmodulin-like proteins in plant?

Calcium signalling, including pulse, amplitude, and duration, is essential for plant development and response to various stimuli. However, the calcium signalling should be decoded and translated by calcium sensors. In plants, three classes of calcium-binding proteins have been identified as calcium sensors, including calcium-dependent protein kinase (CDPK), calcineurin B-like protein (CBL), and calmodulin (CaM). Calmodulin-like proteins (CMLs), which have several EF-hands, also serve as specific calcium sensors and can sense, bind, and interpret the calcium signal during the plant's growth and defense decision-making processes. In recent decades, the function of CMLs in plant development and response to various stimuli has been systematically reviewed, shedding light on the molecular mechanism of plant CML-mediated networks in calcium signal transduction. Here, by providing an overview of CML expression and biological function in plants, we demonstrate that growth-defense trade-offs occur during calcium sensing, an aspect that has not been well studied in recent years.

Experiment and Simulation Reveal Residue Details for How Target Binding Tunes Calmodulin's Calcium-Binding Properties.

We aim to elucidate the molecular mechanism of the reciprocal relation of calmodulin's (CaM) target binding and its affinity for calcium ions (Ca2+), which is central to decoding CaM-dependent Ca2+ signaling in a cell. We employed stopped-flow experiments and coarse-grained molecular simulations that learn the coordination chemistry of Ca2+ in CaM from first-principle calculations. The associative memories as part of the coarse-grained force fields built on known protein structures further influence CaM's selection of its polymorphic target peptides in the simulations. We modeled the peptides from the Ca2+/CaM-binding domain of Ca2+/CaM-dependent kinase II (CaMKII), CaMKIIp (293-310) and selected distinctive mutations at the N-terminus. Our stopped-flow experiments have shown that the CaM's affinity for Ca2+ in the bound complex of Ca2+/CaM/CaMKIIp decreased significantly when Ca2+/CaM bound to the mutant peptide (296-AAA-298) compared to that bound to the wild-type peptide (296-RRK-298). The coarse-grained molecular simulations revealed that the 296-AAA-298 mutant peptide destabilized the structures of Ca2+-binding loops at the C-domain of CaM (c-CaM) due to both loss of electrostatic interactions and differences in polymorphic structures. We have leveraged a powerful coarse-grained approach to advance a residue-level understanding of the reciprocal relation in CaM, that could not be possibly achieved by other computational approaches.

Structures of calmodulin-melittin complexes show multiple binding modes lacking classical anchoring interactions.

Calmodulin (CaM) is a Ca2+ sensor protein found in all eukaryotic cells that regulates a large number of target proteins in a Ca2+ concentration-dependent manner. As a transient-type hub protein, it recognizes linear motifs of its targets, though for the Ca2+-dependent binding, no consensus sequence was identified. Its complex with melittin, a major component of bee venom, is often used as a model system of protein-protein complexes. Yet, the structural aspects of the binding are not well understood, as only diverse, low-resolution data are available concerning the association. We present the crystal structure of melittin in complex with Ca2+-saturated CaMs from two, evolutionarily distant species, Homo sapiens and Plasmodium falciparum, representing three binding modes of the peptide. Results-augmented by molecular dynamics simulations-indicate that multiple binding modes can exist for CaM-melittin complexes, as an intrinsic characteristic of the binding. While the helical structure of melittin remains, swapping of its salt bridges and partial unfolding of its C-terminal segment can occur. In contrast to the classical way of target recognition by CaM, we found that different sets of residues can anchor at the hydrophobic pockets of CaM, which were considered as main recognition sites. Finally, the nanomolar binding affinity of the CaM-melittin complex is created by an ensemble of arrangements of similar stability-tight binding is achieved not by optimized specific interactions but by simultaneously satisfying less optimal interaction patterns in co-existing different conformers.

Binding by calmodulin is coupled to transient unfolding of the third FF domain of Prp40A.

Human pre-mRNA processing protein 40 homolog A (hPrp40A) is a splicing factor that interacts with the Huntington's disease protein huntingtin (Htt). Evidence has accumulated that both Htt and hPrp40A are modulated by the intracellular Ca2+ sensor calmodulin (CaM). Here we report characterization of the interaction of human CM with the third FF domain (FF3 ) of hPrp40A using calorimetric, fluorescence and structural approaches. Homology modeling, differential scanning calorimetry and small angle X-ray scattering (SAXS) data show FF3 forms a folded globular domain. CaM was found to bind FF3 in a Ca2+ -dependent manner with a 1:1 stoichiometry and a dissociation constant (Kd ) of 25 ± 3 µM at 25°C. NMR studies showed that both domains of CaM are engaged in binding and SAXS analysis of the FF3 -CaM complex revealed CaM occupies an extended configuration. Analysis of the FF3 sequence showed that the anchors for CaM binding must be buried in its hydrophobic core, suggesting that binding to CaM requires unfolding of FF3 . Trp anchors were proposed based on sequence analysis and confirmed by intrinsic Trp fluorescence of FF3 upon binding of CaM and substantial reductions in affinity for Trp-Ala FF3 mutants. The consensus model of the complex showed that binding to CaM binding occurs to an extended, non-globular state of the FF3 , consistent with coupling to transient unfolding of the domain. The implications of these results are discussed in the context of the complex interplay of Ca2+ signaling and Ca2+ sensor proteins in modulating Prp40A-Htt function.

Life-threatening arrhythmogenic CaM mutations disrupt CaM binding to a distinct RyR2 CaM-binding pocket.

Calmodulin (CaM) modulates the activity of several proteins that play a key role in excitation-contraction coupling (ECC). In cardiac muscle, the major binding partner of CaM is the type-2 ryanodine receptor (RyR2) and altered CaM binding contributes to defects in sarcoplasmic reticulum (SR) calcium (Ca2+) release. Many genetic studies have reported a series of CaM missense mutations in patients with a history of severe arrhythmogenic cardiac disorders. In the present study, we generated four missense CaM mutants (CaMN98I, CaMD132E, CaMD134H and CaMQ136P) and we used a CaM-RyR2 co-immunoprecipitation and a [3H]ryanodine binding assay to directly compare the relative RyR2-binding of wild type and mutant CaM proteins and to investigate the functional effects of these CaM mutations on RyR2 activity. Furthermore, isothermal titration calorimetry (ITC) experiments were performed to investigate and compare the interactions of the wild-type and mutant CaM proteins with various synthetic peptides located in the well-established RyR2 CaM-binding region (3584-3602aa), as well as another CaM-binding region (4255-4271aa) of human RyR2. Our data revealed that all four CaM mutants displayed dramatically reduced RyR2 interaction and defective modulation of [3H]ryanodine binding to RyR2, regardless of LQTS or CPVT association. Moreover, our isothermal titration calorimetry ITC data suggest that RyR2 3584-3602aa and 4255-4271aa regions interact with significant affinity with wild-type CaM, in the presence and absence of Ca2+, two regions that might contribute to a putative intra-subunit CaM-binding pocket. In contrast, screening the interaction of the four arrhythmogenic CaM mutants with two synthetic peptides that correspond to these RyR2 regions, revealed disparate binding properties and signifying differential mechanisms that contribute to reduced RyR2 association.

Structural Analysis and Diversity of Calmodulin-Binding Domains in Membrane and Intracellular Ca2+-ATPases.

The plasma membrane and autoinhibited Ca2+-ATPases contribute to the Ca2+ homeostasis in a wide variety of organisms. The enzymatic activity of these pumps is stimulated by calmodulin, which interacts with the target protein through the calmodulin-binding domain (CaMBD). Most information about this region is related to all calmodulin modulated proteins, which indicates general chemical properties and there is no established relation between Ca2+ pump sequences and taxonomic classification. Thus, the aim of this study was to perform an in silico analysis of the CaMBD from several Ca2+-ATPases, in order to determine their diversity and to detect specific patterns and amino acid selection in different species. Patterns related to potential and confirmed CaMBD were detected using sequences retrieved from the literature. The occurrence of these patterns was determined across 120 sequences from 17 taxonomical classes, which were analyzed by a phylogenetic tree to establish phylogenetic groups. Predicted physicochemical characteristics including hydropathy and net charge were calculated for each group of sequences. 22 Ca2+-ATPases sequences from animals, unicellular eukaryotes, and plants were retrieved from bioinformatic databases. These sequences allow us to establish the Patterns 1(GQILWVRGLTRLQTQ), 3(KNPSLEALQRW), and 4(SRWRRLQAEHVKK), which are present at the beginning of putative CaMBD of metazoan, parasites, and land plants. A pattern 2 (IRVVNAFR) was consistently found at the end of most analyzed sequences. The amino acid preference in the CaMBDs changed depending on the phylogenetic groups, with predominance of several aliphatic and charged residues, to confer amphiphilic properties. The results here displayed show a conserved mechanism to contribute to the Ca2+ homeostasis across evolution and may help to detect putative CaMBDs.

Hydrophobic interactions within the C terminus pole helices tunnel regulate calcium-dependent inactivation of TRPC3 in a calmodulin-dependent manner.

Recent structural studies have shown that the carboxyl-terminus of many TRP channels, including TRPC3, are folded into a horizontal rib helix that is connected to the vertical pole helix, which play roles in inter-structural interactions and multimerization. In a previous work we identified I807 located in the pole helix with a role in regulation of TRPC3 by STIM1 (Lee et al., 2014, Liu et al., 2022). To further determine the role of the pole helix in TRPC3 function, here we identified key hydrophobic residues in the pole helix that form tight tunnel-like structure and used mutations to probe their role in TRPC3 regulation by Ca2+ and Calmodulin. Our findings suggest that the hydrophobic starch formed by the I807-L818 residues has several roles, it modulates gating of TRPC3 by Ca2+, affects channel selectivity and the channel Ca2+ permeability. Mutations of I807, I811, L814 and L818 all attenuated the Ca2+-dependent inactivation (CDI) of TRPC3, with I807 having the most prominent effect. The extent of modulation of the CDI depended on the degree of hydrophobicity of I807. Moreover, the TRPC3(I807S) mutant showed altered channel monovalent ion selectivity and increased Ca2+ permeability, without affecting the channel permeability to Mg2+ and Ba2+ and without changing the pore diameter. The CDI of TRPC3 was reduced by an inactive calmodulin mutant and by a pharmacological inhibitor of calmodulin, which was eliminated by the I807S mutation. Notably, deletion of STIM1 caused similar alteration of TRPC3 properties. Taken together, these findings reveal a role of the pole helix in CDI, in addition to its potential role in channel multimerization that required gating of TRPC3 by STIM1. Since all TRPC and most TRP channels have pole helix structures, our findings raise the possibility that the pole helix may have similar roles in all the TRP family.

Calmodulin Modulation of Insect Ryanodine Receptors.

Ryanodine receptor (RyR) is a giant calcium release channel located on the membrane of the endoplasmic reticulum (ER). Here, we report the regulation of RyRs from two major agricultural pests, diamondback moth and fall armyworm, by insect calmodulin (CaM). The recombinantly expressed full-length insect RyR could be pulled down by insect CaM in the presence of Ca2+, but the efficiency is lower compared to rabbit RyR1 and insect RyR with the CaM-binding domain (CaMBD) replaced by rabbit RyR1 sequence. Interestingly, the enhanced binding of CaM in the mutant insect RyR resulted in an increased sensitivity to the diamide insecticide chlorantraniliprole (CHL), suggesting that this CaM-CaMBD interface could be targeted by potential synergists acting as molecular glue. The thermodynamics of the binding between insect CaM and CaMBD was characterized by isothermal titration calorimetry, and the key residues responsible for the insect-specific regulation were identified through mutagenesis studies.

Optimized Coordination of Uranyl in Engineered Calmodulin Site 1 Provides a Subnanomolar Affinity for Uranyl and a Strong Uranyl versus Calcium Selectivity.

As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log KpH7 = 9.55 ± 0.11 (KdpH7 = 280 ± 60 pM). Structural analysis by FTIR, XAS, and molecular dynamics simulations suggests an optimized coordination of the CaMΔ-uranyl complex involving bidentate and monodentate carboxylate groups in the uranyl equatorial plane. The main role of this coordination sphere in reaching subnanomolar dissociation constants for uranyl is supported by similar uranyl affinities obtained in a cyclic peptide reproducing CaMΔ binding loop. In addition, CaMΔ presents a uranyl/calcium selectivity of 107 that is even higher in the cyclic peptide.