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1.
Phys Chem Chem Phys ; 22(3): 1092-1096, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31894209

RESUMO

Mechanical force plays a critical role in the relationship between protein structure and function. Force manipulation by Atomic Force Microscope can be significant and trigger chemical and biological activities of proteins. Previously we have reported that Apo-CaM undergoes through a spontaneous tertiary structural rupture under a piconewton compressive force. Here we have observed that the ruptured Apo-CaM molecules can be available to bind with C28W peptide, a typical protein signalling activity that only a Ca2+-activated CaM has. This behaviour is both unexpected and profound, as CaM in its Ca2+-non-activated form has a closed structure which does not presumably allow the molecule to bind to target peptides. In this experiment, we demonstrate that both chemical activation and force activation can play a vital role in biology, such as the cell-signalling protein dynamics and function.


Assuntos
Calmodulina/química , Proteínas/química , Transdução de Sinais , Fenômenos Biomecânicos , Ligação Proteica , Estrutura Terciária de Proteína
2.
J Chem Theory Comput ; 15(12): 6752-6759, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31647864

RESUMO

Free energy landscapes provide insights into conformational ensembles of biomolecules. In order to analyze these landscapes and elucidate mechanisms underlying conformational changes, there is a need to extract metastable states with limited noise. This has remained a formidable task, despite a plethora of existing clustering methods. We present InfleCS, a novel method for extracting well-defined core states from free energy landscapes. The method is based on a Gaussian mixture free energy estimator and exploits the shape of the estimated density landscape. The core states that naturally arise from the clustering allow for detailed characterization of the conformational ensemble. The clustering quality is evaluated on three toy models with different properties, where the method is shown to consistently outperform other conventional and state-of-the-art clustering methods. Finally, the method is applied to a temperature enhanced molecular dynamics simulation of Ca2+-bound Calmodulin. Through the free energy landscape, we discover a pathway between a canonical and a compact state, revealing conformational changes driven by electrostatic interactions.


Assuntos
Calmodulina/química , Entropia , Simulação de Dinâmica Molecular , Análise por Conglomerados , Estrutura Molecular , Eletricidade Estática
3.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505788

RESUMO

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions-N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member-the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.


Assuntos
Calmodulina/química , Modelos Moleculares , Proteínas S100/química , Canais de Cátion TRPM/química , Humanos , Domínios Proteicos
4.
Phys Chem Chem Phys ; 21(36): 19795-19804, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31482888

RESUMO

Allostery plays important roles in the regulation of many biological processes, such as signal transduction and transcriptional regulation. Although great advances have been achieved in understanding the allosteric mechanism through experimental and theoretical investigations, the details of the allosteric process are still not clear. Here, using the N-terminal domain of calmodulin (nCaM) as the model protein, we reported the atomic level characterization of the allosteric process induced by Ca2+ binding through extensive and unbiased molecular dynamics simulations. In two trajectories, it was found that Ca2+ first binds to EF-hand 2 and then induces the conformational transformation of nCaM from the Apo to Holo state assisted by second Ca2+ binding to EF-hand 1 completely. The binding order was consistent with a recent experimental result. The simulations also indicated that the two EF-hands changed conformations synergistically and the EF-hand 2 showed an earlier and more gradual conformational transition. Meanwhile, the allosteric process of nCaM triggered by Ca2+ binding might be completed within hundreds of nanoseconds in a two-state-like manner. This was validated by biased simulations, in which the Ca2+ ions were restrained near the binding sites. This work provides the molecular details of the conformational transition of nCaM triggered by Ca2+ binding.


Assuntos
Cálcio/química , Calmodulina/química , Íons/química , Simulação de Acoplamento Molecular , Domínios Proteicos , Ligação Proteica , Conformação Proteica
5.
Phys Chem Chem Phys ; 21(38): 21213-21222, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31418759

RESUMO

Trivalent actinides and their lanthanide homologues are being scrutinized for their potential health risk when ingested as a result of a range of industrial activities such as mining. Importantly, these ions are known to exhibit high affinity towards calmodulin (CaM). In case of their inadvertent uptake, the holoproteins that are occupied by these cations may block signal transduction pathways or increase the concentration of these ions in intact cells, which could lead to accumulation in human organs. Accordingly, this investigation employed spectroscopy, computational chemistry, calorimetry, and biochemistry to study the results of metal ion substitution on the protein structure, enzymatic activity and chemo- and cytotoxicity of An3+/Ln3+ ions. As will be demonstrated herein, our data confirm the higher affinity of Cm3+ and Eu3+ compared to Ca2+ to all 4 binding sites of CaM, with one site differing from the remaining three. This higher-affinity site will complex Eu3+ in an exothermic fashion; in contrast, ion binding to the three lower-affinity EF-hands was found to be endothermic. The overall endothermic binding process is ascribed to the loss of the hydration shells of the trivalent ions upon protein binding. These findings are supported by extensive quantum chemical calculations of full holo-CaM, which were performed at the MP2 level using the fragment molecular orbital method. The exceptional binding site (EF-hand 3) features fewer negatively charged residues compared to the other EF-hands, thereby allowing Eu3+ and Cm3+ to carry one or two additional waters compared to Ca2+-CaM, while also causing the structure of Cm3+/Eu3+-CaM to become slightly disordered. Moreover, the enzymatic activity decreases somewhat in comparison to Ca2+-CaM. By utilizing a combination of techniques, we were able to generate a comprehensive picture of the CaM-actinide/lanthanide system from the molecular level to its functional impact. Such knowledge could also be applied to other metal-binding proteins.


Assuntos
Calmodulina/química , Calmodulina/metabolismo , Cúrio/química , Európio/química , Sítios de Ligação , Cálcio/química , Cátions , Simulação de Dinâmica Molecular , Conformação Proteica , Água
6.
Molecules ; 24(14)2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319525

RESUMO

The functionalization of VHHs enables their application in almost every aspect of biomedical inquiry. Amino modification remains a common strategy for protein functionalization, though is considered to be inferior to site-specific methods and cause protein property changes. In this paper, four anti-ß2M VHHs were selected and modified on the amino group by NHS-Fluo. The impacts of amino modification on these VHHs were drastically different, and among all th examples, the modified NB-1 maintained the original stability, bioactivity and homogeneity of unmodified NB-1. Specific recognition of VHHs targeting ß2M detected by fluorescence imaging explored the possible applications of VHHs. Via this study, we successfully functionalized the anti-ß2M VHHs through amino modification and the results are able to instruct the simple and fast functionalization of VHHs in biomedical researches.


Assuntos
Epitopos/química , Anticorpos de Domínio Único/isolamento & purificação , Microglobulina beta-2/química , Calmodulina/química , Calmodulina/imunologia , Epitopos/imunologia , Humanos , Imagem Óptica , Estabilidade Proteica , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Microglobulina beta-2/imunologia
7.
Nanoscale ; 11(28): 13397-13406, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31276143

RESUMO

Nanomaterials are ideal for electrochemical biosensors, with their nanoscale dimensions enabling the sensitive probing of biomolecular interactions. In this study, we compare field-effect transistors (FET) comprised of unsorted (un-) and semiconducting-enriched (sc-) single-walled carbon nanotubes (SWCNTs). un-SWCNTs have both metallic and semiconducting SWCNTs in the ensemble, while sc-SWCNTs have a >99.9% purity of semiconducting nanotubes. Both SWCNT FET devices were decorated with gold nanoparticles (AuNPs) and were then employed in investigating the Ca2+-induced conformational change of calmodulin (CaM) - a vital process in calcium signal transduction in the human body. Different biosensing behavior was observed from FET characteristics of the two types of SWCNTs, with sc-SWCNT FET devices displaying better sensing performance with a dynamic range from 10-15 M to 10-13 M Ca2+, and a lower limit of detection at 10-15 M Ca2+.


Assuntos
Cálcio/química , Calmodulina/química , Ouro/química , Nanopartículas Metálicas/química , Nanotubos de Carbono/química , Transistores Eletrônicos , Células HEK293 , Humanos , Conformação Proteica
8.
Fungal Biol ; 123(7): 489-496, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31196518

RESUMO

To develop an antimicrobial agent for preventing the devasting damage caused by rice blast, a novel peptide aptamer was identified to interact with calmodulin (CaM) for the inhibition of the spore development in the pathogen Magnaporthe oryzae. A peptide aptamer designated as SNP-D4, consisted of the scaffold protein Staphylococcus aureus nuclease (SN) and an exposed surface loop of 16 random amino acids, was screened from the constructed peptide aptamer libraries by bacterial two-hybrid system using CaM of M. oryzae as the bait. The preliminary inhibition in the sporulation development was observed after treating with the crude extracts expressing SNP-D4. The inhibition efficacies of the purified SNP-D4 were quantified at the stages of conidial germination, germ tube elongation, and appressorium formation in M. oryzae. The binding affinity analysis revealed that SNP-D4 interacted with CaM at a dissociation constant (Kd) of about 20 µM. Moreover, the N-terminus of CaM was identified as the key binding region.


Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aptâmeros de Peptídeos/metabolismo , Aptâmeros de Peptídeos/farmacologia , Calmodulina/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/efeitos dos fármacos , Sequência de Aminoácidos , Antifúngicos/química , Aptâmeros de Peptídeos/química , Sítios de Ligação , Calmodulina/química , Proteínas Fúngicas/química , Magnaporthe/crescimento & desenvolvimento , Oryza/microbiologia , Biblioteca de Peptídeos , Doenças das Plantas/microbiologia , Ligação Proteica , Engenharia de Proteínas , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento
9.
Nat Commun ; 10(1): 2784, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239443

RESUMO

CaMKIIα plays an essential role in decoding Ca2+ signaling in spines by acting as a leaky Ca2+ integrator with the time constant of several seconds. However, the mechanism by which CaMKIIα integrates Ca2+ signals remains elusive. Here, we imaged CaMKIIα-CaM association in single dendritic spines using a new FRET sensor and two-photon fluorescence lifetime imaging. In response to a glutamate uncaging pulse, CaMKIIα-CaM association increases in ~0.1 s and decays over ~3 s. During repetitive glutamate uncaging, which induces spine structural plasticity, CaMKIIα-CaM association did not show further increase but sustained at a constant level. Since CaMKIIα activity integrates Ca2+ signals over ~10 s under this condition, the integration of Ca2+ signal by CaMKIIα during spine structural plasticity is largely due to Ca2+/CaM-independent, autonomous activity. Based on these results, we propose a simple kinetic model of CaMKIIα activation in dendritic spines.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espinhas Dendríticas/enzimologia , Animais , Cálcio/química , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Calmodulina/química , Calmodulina/metabolismo , Espinhas Dendríticas/genética , Ativação Enzimática , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL
10.
Science ; 364(6442): 787-792, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31123136

RESUMO

Enzymes with a protein kinase fold transfer phosphate from adenosine 5'-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the Legionella meta-effector SidJ adopts a protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the γ-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful Legionella replication in a viable eukaryotic host cell.


Assuntos
Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , Ácido Poliglutâmico/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Fatores de Virulência/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Calmodulina/química , Calmodulina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Células HEK293 , Humanos , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Fosforilação , Ácido Poliglutâmico/química , Ácido Poliglutâmico/genética , Domínios Proteicos/genética , Proteínas Quinases/química , Proteínas Quinases/genética , Ubiquitina-Proteína Ligases/genética , Fatores de Virulência/química , Fatores de Virulência/genética
11.
Chemphyschem ; 20(14): 1860-1868, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31054266

RESUMO

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.


Assuntos
Calmodulina/química , Calmodulina/metabolismo , Calmodulina/genética , Extratos Celulares/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Gadolínio/química , Células HeLa , Humanos , Mutação , Conformação Proteica , Marcadores de Spin
12.
Nat Commun ; 10(1): 1514, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944319

RESUMO

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Modelos Moleculares , Músculo Esquelético/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas
13.
Biomol NMR Assign ; 13(2): 275-278, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30875027

RESUMO

Vitamin A is a necessary nutrient for all mammals, and it is required for the transcription of many genes and vital for vision. While fasting, the vitamin A alcohol form (Retinol) from storage in the liver is mobilized and transported through the bloodstream while bound to retinol binding protein (RBP). Details of how exactly vitamin A is released from RBP and taken into the cells are still unclear. As part of the effort to elucidate the specifics of this process, single-particle cryo-electron microscopy structural studies of STRA6 (the RBP receptor 75-kDa transmembrane receptor protein) were recently reported by Chen et al. (Science, https://doi.org/10.1126/science.aad8266 , 2016). Interestingly, STRA6 from zebrafish was shown to be a stable dimer and bound to calmodulin (CaM), forming a 180-kDa complex. The topology of the STRA6 complex includes 18 transmembrane helices (nine per protomer) and two long horizontal intramembrane helices interacting at the dimer core (Chen et al., in Science, https://doi.org/10.1126/science.aad8266 , 2016). CaM was shown to interact with three regions of STRA6, termed CaMBP1, CaMBP2, and CaMBP3, with the most extensive interactions involving CaMBP2. To further our understanding of Ca2+-dependence of CaM-STRA6 complex formation, studies of the structure and dynamic properties of the CaMBP2-CaM complex were initiated. For this, the 1HN, 13C, and 15N backbone resonance assignments of the 148 amino acid Ca2+-bound calmodulin protein bound to the 27-residue CaMBP2 peptide derived from STRA6 were completed here using heteronuclear multidimensional NMR spectroscopy.


Assuntos
Calmodulina/química , Calmodulina/metabolismo , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/metabolismo , Humanos , Ligação Proteica
14.
Int J Mol Sci ; 20(6)2019 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-30909616

RESUMO

Ca2+ is a universal second messenger that plays a pivotal role in diverse signaling mechanisms in almost all life forms. Since the evolution of life from an aquatic to a terrestrial environment, Ca2+ signaling systems have expanded and diversified enormously. Although there are several Ca2+ sensing molecules found in a cell, EF-hand containing proteins play a principal role in calcium signaling event in plants. The major EF-hand containing proteins are calmodulins (CaMs), calmodulin like proteins (CMLs), calcineurin B-like (CBL) and calcium dependent protein kinases (CDPKs/CPKs). CaMs and CPKs contain calcium binding conserved D-x-D motifs in their EF-hands (one motif in each EF-hand) whereas CMLs contain a D-x3-D motif in the first and second EF-hands that bind the calcium ion. Calcium signaling proteins form a complex interactome network with their target proteins. The CMLs are the most primitive calcium binding proteins. During the course of evolution, CMLs are evolved into CaMs and subsequently the CaMs appear to have merged with protein kinase molecules to give rise to calcium dependent protein kinases with distinct and multiple new functions. Ca2+ signaling molecules have evolved in a lineage specific manner with several of the calcium signaling genes being lost in the monocot lineage.


Assuntos
Motivos de Aminoácidos , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Evolução Molecular , Proteínas de Plantas/química , Plantas/genética , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Relação Estrutura-Atividade
15.
Cell Mol Life Sci ; 76(12): 2299-2328, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30877334

RESUMO

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


Assuntos
Calmodulina/química , Motivos EF Hand , Proteínas/química , Actinina/química , Actinina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calcineurina/química , Calcineurina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Calmodulina/metabolismo , Calpaína/química , Calpaína/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas/metabolismo
16.
J Biol Chem ; 294(15): 6142-6156, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30770467

RESUMO

In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.


Assuntos
Brachypodium , Cálcio , Calmodulina , Transportadores de Ânions Orgânicos , Proteínas de Plantas , Estômatos de Plantas , Substituição de Aminoácidos , Brachypodium/química , Brachypodium/genética , Brachypodium/metabolismo , Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Ativação do Canal Iônico/fisiologia , Malatos/química , Malatos/metabolismo , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estômatos de Plantas/química , Estômatos de Plantas/genética , Estômatos de Plantas/metabolismo
17.
J Chem Theory Comput ; 15(4): 2719-2733, 2019 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-30810317

RESUMO

A new version of the coarse-grained (CG) SIRAH force field for proteins has been developed. Modifications to bonded and non-bonded interactions on the existing molecular topologies significantly ameliorate the structural description and flexibility of a non-redundant set of proteins. The SIRAH 2.0 force field has also been ported to the popular simulation package AMBER, which along with the former implementation in GROMACS expands significantly the potential range of users and performance of this CG force field on CPU/GPU codes. As a non-trivial example of its application, we undertook the structural and dynamical analysis of the most abundant and conserved calcium-binding protein, calmodulin (CaM). CaM is composed of two calcium-binding motifs called EF-hands, which in the presence of calcium specifically recognize a cognate peptide by embracing it. CG simulations of CaM bound to four calcium ions in the presence or absence of a binding peptide (holo and apo forms, respectively) resulted in good and stable ion coordination. The simulation of the holo form starting from an experimental structure sampled near-native conformations, retrieving quasi-atomistic precision. Removing the binding peptide enabled the EF-hands to perform large reciprocal movements, comparable to those observed in NMR structures. On the other hand, the isolated peptide starting from the helical conformation experienced spontaneous unfolding, in agreement with previous experimental data. However, repositioning the peptide in the neighborhood of one EF-hand not only prevented the peptide from unfolding but also drove CaM to a fully bound conformation, with both EF-hands embracing the cognate peptide, resembling the experimental holo structure. Therefore, SIRAH 2.0 shows the capacity to handle a number of structurally and dynamically challenging situations, including metal ion coordination, unbiased conformational sampling, and specific protein-peptide recognition.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Software , Animais , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Bases de Dados de Proteínas , Motivos EF Hand , Humanos , Ligação Proteica , Conformação Proteica , Proteínas/metabolismo
18.
Biomol NMR Assign ; 13(1): 233-237, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30788773

RESUMO

Calcium-dependent inactivation (CDI) of neuronal voltage-gated Ca2+ channels (CaV1.2) is important for synaptic plasticity, which is associated with learning and memory. The Ca2+-dependent binding of calmodulin (CaM) to CaV1.2 is essential for CDI. Here we report NMR assignments for a CaM mutant (D21A/D23A/D25A/E32Q/D57A/D59A/N61A/E68Q, called CaMEF12) that contains two Ca2+ bound at the third and fourth EF-hands (EF3 and EF4) and is bound to the IQ-motif (residues 1644-1665) from CaV1.2 (BMRB accession no. 27692).


Assuntos
Canais de Cálcio/química , Cálcio/metabolismo , Calmodulina/química , Ressonância Magnética Nuclear Biomolecular , Amidas/química , Motivos de Aminoácidos , Humanos , Ligação Proteica , Estrutura Secundária de Proteína
19.
J Biol Chem ; 294(15): 6094-6112, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30808708

RESUMO

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.


Assuntos
Cálcio/química , Calmodulina/química , Canais de Potássio KCNQ/química , Animais , Células CHO , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cricetulus , Cristalografia por Raios X , Humanos , Ativação do Canal Iônico , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
20.
Methods Mol Biol ; 1929: 157-186, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710273

RESUMO

A multitude of Ca2+-sensor proteins containing the specific Ca2+-binding motif (helix-loop-helix, called EF-hand) are of major clinical relevance in a many human diseases. Measurements of troponin, the first intracellular Ca-sensor protein to be discovered, is nowadays the "gold standard" in the diagnosis of patients with acute coronary syndrome (ACS). Mutations have been identified in calmodulin and linked to inherited ventricular tachycardia and in patients affected by severe cardiac arrhythmias. Parvalbumin, when introduced into the diseased heart by gene therapy to increase contraction and relaxation speed, is considered to be a novel therapeutic strategy to combat heart failure. S100 proteins, the largest subgroup with the EF-hand protein family, are closely associated with cardiovascular diseases, various types of cancer, inflammation, and autoimmune pathologies. The intention of this review is to summarize the clinical importance of this protein family and their use as biomarkers and potential drug targets, which could help to improve the diagnosis of human diseases and identification of more selective therapeutic interventions.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Motivos EF Hand , Diagnóstico Precoce , Humanos , Família Multigênica , Parvalbuminas/química , Parvalbuminas/genética , Parvalbuminas/metabolismo , Prognóstico , Proteínas S100/química , Proteínas S100/genética , Proteínas S100/metabolismo , Troponina/química , Troponina/genética , Troponina/metabolismo
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