Calmodulin Peptide Research Articles

Investigation of the Plutonium(IV) Interactions with Two Variants of the EF-Hand Ca-Binding Site I of Calmodulin.

Due to its presence in the nuclear industry and its strong radiotoxicity, plutonium is an actinide of major interest in the event of internal contamination. To improve the understanding of its mechanisms of transport and accumulation in the body, the complexation of Pu(IV) to the most common protein calcium-binding motif found in cells, the EF-hand motif of calmodulin, was investigated. Visible and X-ray absorption spectroscopies (XAS) in solution made it possible to investigate the speciation of plutonium at physiological pH (pH 7.4) and pH 6 in two variants of the calmodulin Ca-binding site I and using Pu(IV) in different media: carbonate, chloride, or nitrate solutions. Three different species of Pu were identified in the samples, with formation of 1:1 Pu(IV):calmodulin peptide complexes, Pu(IV) reduction, and formation of peptide-mediated Pu(IV) hexanuclear cluster.

Population Dynamics of Stretching Excitations of p-Azido-phenylalanine Incorporated in Calmodulin-Peptide Complexes.

We genetically incorporated the unnatural amino acid p-azido-phenylalanine (AzF) into the ubiquitous Ca2+ sensor protein calmodulin (CaM) in complex with different peptides to explore the response of the azido stretching line shape to varying binding motifs with femtosecond infrared spectroscopy. The dynamic response of the azido stretching mode varies in different CaM-peptide complexes. We model these dynamics as coherent excitations of Fermi resonances and extract a lifetime of the azido stretching vibration of about 1 ps. The resulting model parameters are commensurate with the linear infrared absorption lineshapes which suggests that the conformation-sensitive vibrational lineshape could be composed of Fermi resonances that differ between the protein-peptide complexes.

Solution of the structure of a calmodulin-peptide complex in a novel configuration from a variably twinned data set.

Structure determination of conformationally variable proteins can prove challenging even when many possible molecular-replacement (MR) search models of high sequence similarity are available. Calmodulin (CaM) is perhaps the best-studied archetype of these flexible proteins: while there are currently ∼450 structures of significant sequence similarity available in the Protein Data Bank (PDB), novel conformations of CaM and complexes thereof continue to be reported. Here, the details of the solution of a novel peptide-CaM complex structure by MR are presented, in which only one MR solution of marginal quality was found despite the use of 120 different search models, an exclusivity enhanced by the presence of a high degree of hemihedral twinning (overall refined twin fraction = 0.43). Ambiguities in the initial MR electron-density maps were overcome by using MR-SAD: phases from the MR partial model were used to identify weak anomalous scatterers (calcium, sulfur and chloride), which were in turn used to improve the phases, automatically rebuild the structure and resolve sequence ambiguities. Retrospective analysis of consecutive wedges of the original data sets showed twin fractions ranging from 0.32 to 0.55, suggesting that the data sets were variably twinned. Despite these idiosyncrasies and obstacles, the data themselves and the final model were of high quality and indeed showed a novel, nearly right-angled conformation of the bound peptide.

Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering.

The calcium-binding protein calmodulin (CaM) directly binds to membrane transport proteins to modulate their function in response to changes in intracellular calcium concentrations. Because CaM recognizes and binds to a wide variety of target sequences, identifying CaM-binding sites is difficult, requiring intensive sequence gazing and extensive biochemical analysis. Here, we describe a straightforward computational script that rapidly identifies canonical CaM-binding motifs within an amino acid sequence. Analysis of the target sequences from high resolution CaM-peptide structures using this script revealed that CaM often binds to sequences that have multiple overlapping canonical CaM-binding motifs. The addition of a positive charge discriminator to this meta-analysis resulted in a tool that identifies potential CaM-binding domains within a given sequence. To allow users to search for CaM-binding motifs within a protein of interest, perform the meta-analysis, and then compare the results to target peptide-CaM structures deposited in the Protein Data Bank, we created a website and online database. The availability of these tools and analyses will facilitate the design of CaM-related studies of ion channels and membrane transport proteins.

Contrast-Matched Small-Angle X-ray Scattering from a Heavy-Atom-Labeled Protein in Structure Determination: Application to a Lead-Substituted Calmodulin–Peptide Complex

The information content in 1-D solution X-ray scattering profiles is generally restricted to low-resolution shape and size information that, on its own, cannot lead to unique 3-D structures of biological macromolecules comparable to all-atom models derived from X-ray crystallography or NMR spectroscopy. Here we show that contrast-matched X-ray scattering data collected on a protein incorporating specific heavy-atom labels in 65% aqueous sucrose buffer can dramatically enhance the power of conventional small- and wide-angle X-ray scattering (SAXS/WAXS) measurements. Under contrast-matching conditions the protein is effectively invisible and the main contribution to the X-ray scattering intensity arises from the heavy atoms, allowing direct extraction of pairwise distances between them. In combination with conventional aqueous SAXS/WAXS data, supplemented by NMR-derived residual dipolar couplings (RDCs) measured in a weakly aligning medium, we show that it is possible to position protein domains relative to one another within a precision of 1 Å. We demonstrate this approach with respect to the determination of domain positions in a complex between calmodulin, in which the four Ca(2+) ions have been substituted by Pb(2+), and a target peptide. The uniqueness of the resulting solution is established by an exhaustive search over all models compatible with the experimental data, and could not have been achieved using aqueous SAXS and RDC data alone. Moreover, we show that the correct structural solution can be recovered using only contrast-matched SAXS and aqueous SAXS/WAXS data.

Interactions of calmodulin with death-associated protein kinase peptides: experimental and modeling studies

We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM–DAPK2 S308D mutant peptide is also reported. The structures of CaM–DAPK peptide complexes present a mode of CaM–kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the largest contribution coming from the Trp305. About two-thirds of the buried surface area corresponds to nonpolar residues, showing that hydrophobic interactions play an important role in these CaM–peptide complexes. The simulation results agree with the experimental data in predicting a small effect of the S308D mutation on CaM interactions with DAPK2, suggesting that this mutation is not a good model for the S308 phosphorylation.

Quantification of Protein−Protein Interactions with Chemical Cross-Linking and Mass Spectrometry

Chemical cross-linking in combination with mass spectrometry has largely been used to study protein structures and protein-protein interactions. Typically, it is used in a qualitative manner to identify cross-linked sites and provide a low-resolution topological map of the interacting regions of proteins. Here, we investigate the capability of chemical cross-linking to quantify protein-protein interactions using a model system of calmodulin and substrates melittin and mastoparan. Calmodulin is a well-characterized protein which has many substrates. Melittin and mastoparan are two such substrates which bind to calmodulin in 1:1 ratios in the presence of calcium. Both the calmodulin-melittin and calmodulin-mastoparan complexes have had chemical cross-linking strategies successfully applied in the past to investigate topological properties. We utilized an excess of immobilized calmodulin on agarose beads and formed complexes with varying quantities of mastoparan and melittin. Then, we applied disuccinimidyl suberate (DSS) chemical cross-linker, digested and detected cross-links through an LC-MS analytical method. We identified five interpeptide cross-links for calmodulin-melittin and three interpeptide cross-links for calmodulin-mastoparan. Using cross-linking sites of calmodulin-mastoparan, we demonstrated that mastoparan also binds in two orientations to calmodulin. We quantitatively demonstrated that both melittin and mastoparan preferentially bind to calmodulin in a parallel fashion, which is opposite to the preferred binding mode of the majority of known calmodulin binding peptides. We also demonstrated that the relative abundances of cross-linked peptide products quantitatively reflected the abundances of the calmodulin peptide complexes formed.

Pinpointing Calmodulin on Functioning KCNQ Channels

Calmodulin (CaM) is a ubiquitous intracellular calcium sensor that associates with and modulates several different classes of ion channel complexes. Although crystallographic studies have provided molecular insight into CaM binding using isolated peptide fragments of ion channels, the location and orientation of CaM bound to fully folded channels in the membrane is unknown. To locate CaM bound to KCNQ2/KCNQ3 (Q2/Q3) K+ channels functioning at the cell surface, we synthesized a panel of derivatized CaM peptides that bind and inhibit K+ channel function. Using this panel of reagents, we have located CaM bound to the Q2/Q3 C-terminus relative to the internal tetraethylammonium blocking site. The application of this novel approach to investigate the location, stoichiometry, and dynamics of CaM binding to a variety of ion channel complexes will be discussed.

Calmodulin association with the synthetic ERα17p peptide investigated by mass spectrometry

Implication of calmodulin ( CaM) in breast cancer development has been proposed, justifying the interest for ER/calmodulin interaction. The association of the synthetic peptide ERα17p (H–PLMIKRSKKNSLALSLT–OH), which corresponds to the known ER interaction site of CaM, was investigated. Under physiologically conditions, 1:1 complex formation was observed using an ESI-ITMS instrument. This equimolar complex with CaM was only formed in presence of calcium. The binding of ERα17p to CaM is probably due to presence of four basic residues (K 299RSKK 303) in its sequence. Its relative binding affinity in solution by ESI-MS was evaluated by performing competitive binding experiments with other peptides more (ERα17pKR: KRSKR) or less basic (ERα17pAA: KRSAA) than ERα17p as well as Melittin (Mel), selected as a reference peptide since it is known to form a high-affinity complex with CaM. Relative affinities of these peptides for CaM were classified in the following decreasing order: Mel > ERα17pKR ∼ ERα17p ≫ ERα17pAA. Interestingly, another ion series showing two peptides for one protein was detected. The specificity of this complex not often reported for Melittin is discussed.

The world is flat

NMR-measured order parameters of methyl groups can be used to quantify the entropy of protein conformational change associated with calmodulin–peptide ligand interactions. This conformational entropy is a major contributor to the affinity of calmodulin interactions and can now be determined experimentally on a per-atom basis.

Biochemical and Functional Characterization of Crystallographic Ca2+/CaM-Cav1.2 A-C-IQ Complex Dimer

The ubiquitous calcium sensor calmodulin (CaM) mediates two important voltage-gated calcium channel (CaV) calcium-dependent modulatory mechanisms through its interaction with the CaV C-terminal tail. Here, we report the structure of Ca2+/CaM bound to a portion of the CaV1.2 C-terminus with three consecutive CaM binding motifs (A-C-IQ domain). The structure reveals two channel chains dimerized via bridging CaMs and interactions between two long, antiparallel helices. Unlike the crystal structure, the CaM peptide complex is a monomer in solution that corresponds to a single channel chain and two CaMs. Disruption of the crystallographic inter-helix interactions had minimal effect on CDI and CDF of full-length CaV1.2. Moreover, subunit counting experiments using CaV1.2-EGFP fusion proteins clearly indicate that CaV1.2 is a monomer in cell membranes. Thus, contrary to previously proposed models, there appears to be no role for dimerization in channel function.

Determinants in CaV1 Channels That Regulate the Ca2+ Sensitivity of Bound Calmodulin

Calmodulin binds to IQ motifs in the alpha(1) subunit of Ca(V)1.1 and Ca(V)1.2, but the affinities of calmodulin for the motif and for Ca(2+) are higher when bound to Ca(V)1.2 IQ. The Ca(V)1.1 IQ and Ca(V)1.2 IQ sequences differ by four amino acids. We determined the structure of calmodulin bound to Ca(V)1.1 IQ and compared it with that of calmodulin bound to Ca(V)1.2 IQ. Four methionines in Ca(2+)-calmodulin form a hydrophobic binding pocket for the peptide, but only one of the four nonconserved amino acids (His-1532 of Ca(V)1.1 and Tyr-1675 of Ca(V)1.2) contacts this calmodulin pocket. However, Tyr-1675 in Ca(V)1.2 contributes only modestly to the higher affinity of this peptide for calmodulin; the other three amino acids in Ca(V)1.2 contribute significantly to the difference in the Ca(2+) affinity of the bound calmodulin despite having no direct contact with calmodulin. Those residues appear to allow an interaction with calmodulin with one lobe Ca(2+)-bound and one lobe Ca(2+)-free. Our data also provide evidence for lobe-lobe interactions in calmodulin bound to Ca(V)1.2.

Mutations in the i3N loop calmodulin (CaM)‐binding domain of 5‐HT1A receptor attenuate ERK phosphorylation in CHO‐K1 cells

Our laboratory previously has shown that calmodulin (CaM) binds to two sites in the 5‐HT1A receptor i3 loop; however, the functional significance of the interaction of CaM with the 5‐HT1A receptor is not clear. Because ERK activation is one of the primary signals of the G‐protein coupled 5‐HT1A receptor, we wanted to determine if a receptor deficient in CaM binding has altered ERK activation. We constructed a double point mutation (R223E/K232E) in the 5‐HT1A receptor, and demonstrated that it was well‐expressed after transfection into CHO cells. It binds normally to two agonists (5‐HT and 8‐OH‐DPAT) and maintains intact coupling of the receptor to G‐proteins as demonstrated by GTP‐sensitive high affinity binding. Dot‐blots with biotinylated CaM‐enzyme‐linked avidin system showed that CaM binds to the wild‐type 5‐HT1A peptide in the presence of Ca2+; in contrast, binding of CaM to [R223E/K232E]5‐HT1Ai3N peptide was greatly attenuated, suggesting that the affinity of the mutated 5‐HT1A peptide for CaM was markedly reduced. We stably transfected CHO‐K1 cells with the wild‐type 5‐HT1A receptor or [R223E/K232E]5‐HT1A receptor construct, and measured ERK phosphorylation over time. ERK phosphorylation peaked between 5 and 10 minutes for the wild‐type 5‐HT1A receptor, and peaked at 5 minutes for the mutated [R223E/K232E]5‐HT1A receptor. Interestingly, ERK phosphorylation was decreased dramatically in CHO‐K1 cells transfected with the [R223E/K232E]5‐HT1A receptor construct when compared with the wild‐type 5‐HT1A receptor. These results strongly support a critical role for direct CaM binding to the 5‐HT1A receptor for effective ERK phosphorylation.

Selective binding of uranyl cation by a novel calmodulin peptide

A 33-amino acid peptide corresponding to the helix-loop-helix motif of the calcium binding site I of the protein calmodulin from Paramecium Tetraurelia has been synthesized its binding properties with heavy metal ions have been studied. Herein, we demonstrate that two mutations of two aspartic acid residues in the peptide sequence gave access to a new peptide, which was selective for the uranyl ion UO2 2+. This new peptide can be useful for the development of selective uranyl biosensors to monitor the presence of uranium in contaminated environments.

Sites on Calmodulin That Interact with the C-terminal Tail of Cav1.2 Channel

Two fragments of the C-terminal tail of the alpha(1) subunit (CT1, amino acids 1538-1692 and CT2, amino acids 1596-1692) of human cardiac L-type calcium channel (Ca(V)1.2) have been expressed, refolded, and purified. A single Ca(2+)-calmodulin binds to each fragment, and this interaction with Ca(2+)-calmodulin is required for proper folding of the fragment. Ca(2+)-calmodulin, bound to these fragments, is in a more extended conformation than calmodulin bound to a synthetic peptide representing the IQ motif, suggesting that either the conformation of the IQ sequence is different in the context of the longer fragment, or other sequences within CT2 contribute to the binding of calmodulin. NMR amide chemical shift perturbation mapping shows the backbone conformation of calmodulin is nearly identical when bound to CT1 and CT2, suggesting that amino acids 1538-1595 do not contribute to or alter calmodulin binding to amino acids 1596-1692 of Ca(V)1.2. The interaction with CT2 produces the greatest changes in the backbone amides of hydrophobic residues in the N-lobe and hydrophilic residues in the C-lobe of calmodulin and has a greater effect on residues located in Ca(2+) binding loops I and II in the N-lobe relative to loops III and IV in the C-lobe. In conclusion, Ca(2+)-calmodulin assumes a novel conformation when part of a complex with the C-terminal tail of the Ca(V)1.2 alpha(1) subunit that is not duplicated by synthetic peptides corresponding to the putative binding motifs.

Tryptophan 1093 Is Largely Responsible for the Slow Off Rate of Calmodulin from Plasma Membrane Ca2+ Pump 4b

Tryptophan 1093 resides in the 28-residue calmodulin-binding/autoinhibitory domain of the plasma membrane Ca(2+) pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was approximately 2-fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K(0.5) of 9 nm for wild type to 144 nm (assayed at 700 nm free Ca(2+)). Pre-steady state measurements of the rate of activation by Ca(2+)-calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the half-time of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca(2+)-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulin-activated pump.

Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin

Isoform 3f of the plasma membrane Ca(2+) pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K(d) of 15 nM) than the corresponding peptide of isoform 2b (K(d) value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.

Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E

The N-terminal domain of human apolipoprotein E3 (apoE3) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its low density lipoprotein receptor recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-apoE3(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A. The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable apolipoproteins and are consistent with the hypothesis that, upon lipid binding, apoE3(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.

Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin.

The binding of Mg2+ to calmodulin (CaM) and the effect of Mg2+ on the binding of Ca2+-CaM to target peptides were examined using two-dimensional nuclear magnetic resonance and fluorescence spectroscopic techniques. We found that Mg2+ preferentially binds to Ca2+-binding sites I and IV of CaM in the absence of Ca2+ and that Ca2+-binding site III displays the lowest affinity for Mg2+. In contrast to the marked structural transitions induced by Ca2+ binding, Mg2+ binding causes only localized conformational changes within the four Ca2+-binding loops of CaM. Therefore, Mg2+ does not seem to be able to cause significant structural effects required for the interaction of CaM with target proteins. The presence of excess Mg2+ (up to 10 mM) does not change the order and cooperativity of Ca2+ binding to CaM, and as expected, the structure of Ca2+-saturated CaM is not affected by the presence of Mg2+. However, we found that the binding of Ca2+-saturated CaM to target peptides is affected by Mg2+ with the binding affinity decreasing as the Mg2+ concentration increases. Three different peptides, corresponding to the CaM binding domain of skeletal muscle myosin light-chain kinase (MLCK), CaM-dependent cyclic nucleotide phosphodiesterase (PDE), and smooth muscle caldesmon (CaD), were examined and show different reductions in their affinities toward CaM. The CaM-binding affinity of the MLCK peptide in the presence of 50 mM Mg2+ is approximately 40-fold lower than that seen in the absence of Mg2+, and a similar response was observed for the PDE peptide. The affinity of the CaD peptide for CaM also shows a Mg2+ dependence, though to a much lower magnitude. The Mg2+-dependent decrease in the affinities between CaM and its target peptides is an intrinsic property of Mg2+ rather than a nonspecific ionic effect, as other metal ions such as Na+ do not completely replicate the effect of Mg2+. The inhibitory effect of Mg2+ on the formation of complexes between CaM and its targets may contribute to the specificity of CaM in target activation in response to cellular Ca2+ concentration fluctuations.

Calmodulin-binding function of LLP segments from the HIV type 1 transmembrane protein is conserved among natural sequence variants.

LLP1 is a peptide, derived from the cytoplasmic tail of HIV-1 TM glycoprotein, that binds and inhibits calmodulin; this region is generally conserved among isolates, but amino acid variation does exist both within clade B and among different clades, as well as SIV. In light of previous studies showing that selected single amino acid changes can have a qualitatively significant effect on the calmodulin-binding properties of this peptide, we sought to examine the properties of naturally occurring variant LLP1 sequences. Using a quantitative fluorescence-based method to measure dissociation constants of calmodulin-LLP1 complexes, a remarkable conservation of calmodulin-binding function among natural variants was revealed. In contrast, engineered nonconservative single amino acid changes altered the affinity of the peptide for calmodulin. The results show that the calmodulin-binding function is well preserved despite the sequence variation observed in nature, suggesting that this region of the TM protein is important to viral replication.