Effect of introducing different carboxylate-containing side chains at position 85 on chromophore formation and proton transport in bacteriorhodopsin.

During the initial stages of the bacteriorhodopsin photocycle, a proton is transferred from the Schiff base to the deprotonated carboxylate of Asp85. Earlier studies have shown that replacement of Asp85 by Asn completely abolishes proton transport activity, whereas extension of the side chain by an additional carbon-carbon bond (Asp85-->Glu) results in a functional proton pump. Here we show that extension of the Asp85 side chain by two additional bond lengths also results in a functional proton pump as long as the terminal group is a carboxylate moiety. These side chains were created by modification of the cysteine residue in the Asp85-->Cys mutant with either iodoacetic acid or iodoacetamide. In vitro chromophore formation studies show that the rate of Schiff base protonation in mutants that contain a carboxylate at residue 85 is invariably faster than in mutants that contain neutral substitutions at this position. We conclude that in bacteriorhodopsin, there is considerable tolerance in the volume of the side chain that can be accommodated at position 85 and that the presence of a carboxylate at residue 85 is important both for proton pumping and for stabilizing the protonated Schiff base.     

Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7: A REGULATOR OF GOLGI TRAFFICKING*

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca²⁺ binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca²⁺ but not Mg²⁺ and undergoes significant conformational changes in both secondary and tertiary structure upon Ca²⁺ binding. The Ca²⁺-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca²⁺-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.     

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^II(Cys112Asp) azurin can be reduced by excess [Ru^II(NH₃)₆]²⁺, resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c ₅₅₁ (μ?=?0.1?M sodium phosphate (pH?7.0);E°(Cu^II/I)?=?180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^II(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^II–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.     

Loss of the calmodulin-dependent inhibition of the RyR1 calcium release channel upon oxidation of methionines in calmodulin

The oxidation of methionines in calmodulin (CaM) can affect the activity of calcium pumps and channels to modulate the amplitude and duration of calcium signals. We have therefore investigated the possible oxidation of CaM in skeletal muscle and its effect on the CaM-dependent regulation of the RyR1 calcium release channel. Taking advantage of characteristic reductions in electrophoretic mobility determined by SDS-PAGE, we find that approximately two methionines are oxidized in CaM from skeletal muscle. The functional effect of CaM oxidation on the open probability of the RyR1 calcium release channel was assessed through measurements of [3H]ryanodine binding using a heavy sarcoplasmic reticulum preparation enriched in RyR1. There is a biphasic regulation of RyR1 by unoxidized CaM, in which calcium-activated CaM acts to enhance the calcium sensitivity of channel closure, while apo-CaM functions to enhance channel activity at resting calcium levels. We find that physiological levels of CaM oxidation preferentially weaken the CaM-dependent inhibition of the RyR1 calcium release channel observed at activating micromolar levels of calcium. In contrast, the oxidation of CaM resulted in minimal functional changes in the CaM-dependent activation of RyR1 at resting nanomolar calcium levels. Oxidation does not significantly affect the high-affinity binding of calcium-activated CaM to the CaM-binding sequence of RyR1; rather, methionine oxidation disrupts interdomain interactions between the opposing domains of CaM in complex with the CaM-binding sequence of RyR1 that normally function as part of a conformational switch associated with RyR1 inhibition. These results suggest that the oxidation of CaM can contribute to observed elevations in intracellular calcium levels in response to conditions of oxidative stress observed during biological aging. We suggest that the sensitivity of RyR1 channel activity to CaM oxidation may function as part of an adaptive cellular response that enhances the duration of calcium transients to promote enhanced contractility.     

Site-specific methionine oxidation in calmodulin affects structural integrity and interaction with Ca2+/calmodulin-dependent protein kinase II

Methionine oxidation in the ubiquitous calcium signaling protein calmodulin (CaM) is known to disrupt downstream signaling and target CaM for proteasomal degradation. The susceptibility of CaM to oxidation in the different conformations that are sampled during calcium signaling is currently not well defined. Using an integrative mass spectrometry (MS) approach, applying both native MS and LC/MS/MS, we unravel molecular details of CaM methionine oxidation in the context of its interaction with the Ca(2+)/CaM-dependent protein kinase II (CaMKII). Sensitivity to methionine oxidation in CaM was found to vary according to the conformational state. Three methionine residues (Met71, 72, 145) show increased reactivity in calcium-saturated CaM (holo-CaM) compared to calcium-free CaM (apo-CaM), which has important consequences for oxidation-targeted proteasomal degradation. In addition, all four methionines in the C-terminal lobe (Met109, 124, 144 and 145) are found to be protected from oxidation in a peptide-based model of the CaMKII-bound conformation (cbp-CaM). We furthermore demonstrate that the oxidation of Met144 and 145 inhibits the interaction of CaM with CaMKII. cbp-CaM, in contrast to apo- and holo-CaM, maintains its ability to bind CaMKII under simulated conditions of oxidative stress and is also protected from oxidation-induced unfolding. Thus, we show that the susceptibility towards oxidation of specific residues in CaM is tightly linked to its signaling state and conformation, which has direct implications for calcium/CaM-CaMKII related signaling.     

Mechanistic and structural analyses of the roles of Arg409 and Asp402 in the reaction of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 A resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.     

A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr¹⁴⁹⁴ and Tyr¹⁴⁹⁵) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.     

Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin

The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different k_on and k_off rates in the presence and absence of Ca²⁺, which could play a role in defining the levels of free CaM during Ca²⁺ transients. The initial goal of the current study was to determine whether Ca²⁺ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca²⁺-binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca²⁺ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca²⁺ binding properties of the N-domain. Conversion of Asp⁹³ to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp⁹³ and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca²⁺, the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture.     

Diastereoselective protein methionine oxidation by reactive oxygen species and diastereoselective repair by methionine sulfoxide reductase

Recent studies have shown that the "calcium-sensor" protein calmodulin (CaM) suffers an age-dependent oxidation of methionine (Met) to methionine sulfoxide (MetSO) in vivo. However, MetSO did not accumulate on the Met residues that show the highest solvent-exposure. Hence, the pattern of Met oxidation in vivo may give hints as to which reactive oxygen species and oxidation mechanisms participate in the oxidation of this important protein. Here, we have exposed CaM under a series of different reaction conditions (pH, [Ca(2+)], [KCl]) to various biologically relevant reactive oxygen species and oxidizing systems (peroxides, HOCl, peroxynitrite, singlet oxygen, metal-catalyzed oxidation, and peroxidase-catalyzed oxidation) to investigate whether one of these systems would lead to an oxidation pattern of CaM similar to that observed in vivo. However, generally, these oxidizing conditions led to a preferred or exclusive oxidation of the C-terminal Met residues, in contrast to the oxidation pattern of CaM observed in vivo. Hence, none of the employed oxidizing conditions was able to mimic the age-dependent oxidation of CaM in vivo, indicating that other, yet unidentified oxidation mechanisms may be important in vivo. Some oxidizing species showed a quite-remarkable diastereoselectivity for the formation of either L-Met-D-SO or L-Met-L-SO. Diastereoselectivity was dependent on the nature of the oxidizing species but was less a function of the location of the target Met residue in the protein. In contrast, diastereoselective reduction of L-Met-D-SO by protein methionine sulfoxide reductase (pMSR) was efficient regardless of the position of the L-Met-D-SO residue in the protein and the presence or absence of calcium. With only the L-Met-D-SO diastereomer being a substrate for pMSR, any preferred formation of L-Met-L-SO in vivo may cause the accumulation of MetSO unless the oxidized protein is substrate for (accelerated) protein turnover.     

Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin

Trifluoperazine (TFP; Stelazine?) is an antagonist of calmodulin (CaM), an essential regulator of calcium‐dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca²⁺)₄‐CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the “open” conformation seen in CaM‐kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca²⁺)₄‐CaM and explore differential effects on the N‐ and C‐domains of CaM, stoichiometric TFP titrations of CaM were monitored by ¹⁵N‐HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca²⁺)₄‐CaM. In both cases, the preferred site was in the C‐domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP‐binding sites in apo CaM appeared distinct from those in (Ca²⁺)₄‐CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ‐motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N‐domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the “closed,” “semi‐open,” and “open” domains of CaM. In physiological processes, apo CaM, as well as (Ca²⁺)₄‐CaM, needs to be considered a potential target of drug action. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Interactions of molybdocene dichloride with histidine- and methionine-containing peptides

The reaction behavior of the antitumor active metallocene dihalide Cp₂MoCl₂ (Cp = η⁵-cyclopentadienyl) towards AcHis, AcHis(1-Me), AcHis(3-Me), His-Gly, AcHis-Gly-His, AcMet, Gly-Met-Gly and cyclo-Met-Met has been studied in solution at 2.5 ? pD ? 7.4 by using ¹H NMR spectroscopy. The histidine-containing substrates were found to bind the Cp₂Mo²⁺ unit through the terminal carboxylate group or through the N1 nitrogen of the imidazole ring, depending on the pD value. At physiological pH, coordination takes place exclusively at the imidazole ring with the percentage of Cp₂Mo²⁺-His adducts in 1:1 mixtures being about 70%. By contrast, the thioether group in the side chain of methionine has a very low affinity for the Cp₂Mo²⁺ group. Monodentate S-coordination could not be detected. For AcMet, binding through the carboxylate group was observed as the only coordination mode, while in the case of Gly-Met-Gly Mo-S interaction occurs in combination with carboxylate coordination leading to a S,O-macrochelate in low yield. Coordination to methionine peptides only takes place at pD ? 6, while at physiological pH interactions with the weak donor sites are not observed due to predominating dimerization of [Cp₂Mo(H₂O)(OH)]⁺ to [Cp₂Mo(μ-OH)₂MoCp₂]²⁺. At c(Cl⁻) = 100 mM competitive Cl⁻ coordination reduces the amount of carboxylate and S,O-coordination significantly, while imidazole coordination is not affected.     

Low-temperature IR spectroscopy reveals four stages of water loss during lyophilization of hen egg white lysozyme

Water loss during lyophilization of a 49.4 mg/mL solution of lysozyme in D₂O was studied with ir spectroscopy using a low-temperature, single reflection, horizontal, attenuated, total reflectance accessory. Four regions of water loss were identified and assignable to different forms of bound water. The amide I band begins to shift to higher frequency while the amide II concurrently shifts to lower frequency and broadens after the first stage of water loss (sublimation) at ?10°C. Additionally, the carboxylate band (at 1584 cm^?1) shifts slightly to lower frequency. A second stage at 17°C is characterized by continued shifts in the carboxylate and amide II bands to low frequency, further broadening in the amide II and greater shift to high frequency in the amide I (ascribed to the removal of periphery water around the protein). At the third stage of water loss, the carboxylate band decreases substantially in relative absorbance (consistent with the removal of water from the carbonyl backbone). In the fourth and last stage, the carboxylate band nearly disappears and water loss is very slow. Based upon a final level of hydration of 0.037 h, the last stage corresponds to 25% completion of the removal of water associated with ionizable side chains. From start to finish, the amide I shifts 9 cm^?1 to higher frequency. © 1994 John Wiley & Sons, Inc.     

Molecular Tuning of an EF-Hand-like Calcium Binding Loop : Contributions of the Coordinating Side Chain at Loop Position 3

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca²⁺ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca²⁺ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca²⁺ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca²⁺ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.     

Crystal structures of α‐dioxygenase from Oryza sativa: Insights into substrate binding and activation by hydrogen peroxide

α‐Dioxygenases (α‐DOX) are heme‐containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α‐DOX oxygenate a variety of 14–20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro‐R hydrogen from the α‐carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H₂O₂). We determined the X‐ray crystal structures of wild type α‐DOX from Oryza sativa, the wild type enzyme in complex with H₂O₂, and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of α‐DOX such that the carboxylate forms ionic interactions with His‐311 and Arg‐559. Thr‐316 aids in the positioning of carbon‐2 for hydrogen abstraction. Twenty‐five of the twenty eight contacts made between PA and residues lining the active site occur within the carboxylate and first eight carbons, indicating that interactions within this region of the substrate are responsible for governing selectivity. Comparison of the wild type and H₂O₂ structures provides insight into enzyme activation. The binding of H₂O₂ at the distal face of the heme displaces residues His‐157, Asp‐158, and Trp‐159 ～2.5 Å from their positions in the wild type structure. As a result, the Oδ2 atom of Asp‐158 interacts with the Ca atom in the calcium binding loop, the side chains of Trp‐159 and Trp‐213 reorient, and the guanidinium group of Arg‐559 is repositioned near Tyr‐379, poised to interact with the carboxylate group of the substrate.     

Redox modulation of cellular signaling and metabolism through reversible oxidation of methionine sensors in calcium regulatory proteins

Adaptive responses associated with environmental stressors are critical to cell survival. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met(144) or Met(145) results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylation-dependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in response to beta-adrenergic signaling in the heart. We suggest that under acute conditions, such as inflammation or ischemia, these types of mechanisms ensure minimal nonspecific cellular damage, allowing for rapid restoration of cellular function through repair of oxidized methionines by methionine sulfoxide reductases and degradation pathways after restoration of normal cellular redox conditions.     

Ligand requirements for Ca2+ binding to EGF-like domains.

Site-specific mutagenesis studies of the first epidermal growth factor-like (EGF-like) domain of human clotting factor IX suggest that the calcium-binding site present in this domain (dissociation constant Kd = 1.8 mM at pH 7.5 and ionic strength I = 0.15) involved the carboxylate residues Asp47, Asp49 and Asp64. To further characterize the ligands required for calcium binding to EGF-like domains, two new mutations, Asp47----Asn and Asp49----Asn, were introduced into the domain by peptide synthesis. 1H-NMR spectroscopy was used to obtain the dissociation constants for calcium binding to these mutations. Calcium binding to the Asp49----Asn modified domain is only mildly affected (Kd = 6 mM, I = 0.15), whereas binding to the Asp47----Asn modified domain is severely reduced (Kd = 42 mM, I = 0.15). From these data, it is proposed that the anionic oxygen atoms of the side chains of residues 47 and 64 are essential for calcium binding, whereas the side chain ligand for calcium at residue 49 can be a carboxyamide oxygen. As a control, the introduction of the modification Glu78----Asp in a region of the domain not believed to be involved in calcium binding had very little effect on the Kd for calcium (Kd = 2.6 mM, I = 0.15). Finally, the effect of an Asp47----Gly substitution found in the natural haemophilia B mutant, factor IXAlabama, was investigated. This peptide has a markedly reduced affinity for calcium (Kd = 37 mM, I = 0.15), suggesting that the defect in factor IXAlabama is due to impaired calcium binding to its first EGF-like domain.     

The Calmodulin-Binding,Short Linear Motif,NSCaTE Is Conserved in L-Type Channel Ancestors of Vertebrate Cav1.2 and Cav1.3 Channels

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca²⁺ concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Ca_v1.1 and Ca_v1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCa_v1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca²⁺-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.     

A point mutation in a plant calmodulin is responsible for its inhibition of nitric-oxide synthase

The calcium/calmodulin-dependent activation of nitric-oxide synthase (NOS) and its production of nitric oxide (NO) play a key regulatory role in plant and animal cell function. SCaM-1 is a plant calmodulin (CaM) isoform that is 91% identical to mammalian CaM (wild type CaM (wtCaM)) and a selective competitive antagonist of NOS (Cho, M. J., Vaghy, P. L., Kondo, R., Lee, S. H., Davis, J. P., Rehl, R., Heo, W. D., and Johnson, J. D. (1998) Biochemistry 37, 15593-15597). We have used site-directed mutagenesis to show that a point mutation, involving the substitution of valine for methionine at position 144, is responsible for SCaM-1's inhibition of mammalian NOS. An M144V mutation in wild type CaM produced a mutant (M144V) which exhibited nearly identical inhibition of NOS's NO production and NADPH oxidation, with a similar K(i) (approximately 15 nM) as SCaM-1. A V144M back mutation in SCaM-1 significantly restored its ability to activate NOS's catalytic functions. The length of the hydrophobic amino acid side chain at position 144 appears to be critical for NOS activation, since M144L and M144F activated NOS while M144V and M144C did not. Despite their competitive antagonism of NOS, M144V, like SCaM-1, exhibited a similar dose-dependent activation of phosphodiesterase and calcineurin as wtCaM. SCaM-1 and M144V produced greater inhibition of NOS's oxygenase domain function (NO production) than its reductase domain functions (NADPH oxidation and cytochrome c reduction). Thus, CaM's methionine 144 plays a critical role the activation of NOS, presumably by influencing the function of NOS's oxygenase domain.     

Functional importance of calcium binding sites in outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca²⁺ as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca²⁺ transfer to the interfacial calcium site.     

Retention of Conformational Entropy upon Calmodulin Binding to Target Peptides Is Driven by Transient Salt Bridges

Calmodulin (CaM) is a highly flexible calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. To identify how binding affects CaM motions, and its relationship to conformational entropy and target peptide sequence, we have employed fully atomistic, explicit solvent molecular dynamics simulations of unbound CaM and CaM bound to five different target peptides. The calculated CaM conformational binding entropies correlate with experimentally derived conformational entropies with a correlation coefficient R² of 0.95. Selected side-chain interactions with target peptides restrain interhelical loop motions, acting to tune the conformational entropy of the bound complex via widely distributed CaM motions. In the complex with the most conformational entropy retention (CaM in complex with the neuronal nitric oxide synthase binding sequence), Lys-148 at the C-terminus of CaM forms transient salt bridges alternating between Glu side chains in the N-domain, the central linker, and the binding target. Additional analyses of CaM structures, fluctuations, and CaM-target interactions illuminate the interplay between electrostatic, side chain, and backbone properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy, and suggest a need to consider conformational dynamics in optimizing binding affinities.