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1.
Nat Commun ; 15(1): 6498, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39090128

RESUMO

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric compounds as therapeutics, an understanding of the functional and structural basis of their effects is limited. Here we use multiple approaches to dissect the functional and structural effects of orthosteric versus allosteric ligands. We find, using electrophysiological and live cell imaging assays, that both agonists and positive allosteric modulators (PAMs) can drive activation and internalization of group II and III mGluRs. The effects of PAMs are pleiotropic, boosting the maximal response to orthosteric agonists and serving independently as internalization-biased agonists across mGluR subtypes. Motivated by this and intersubunit FRET analyses, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling.


Assuntos
Microscopia Crioeletrônica , Receptores de Glutamato Metabotrópico , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/agonistas , Regulação Alostérica , Humanos , Células HEK293 , Ligantes , Animais , Transferência Ressonante de Energia de Fluorescência , Domínios Proteicos
2.
bioRxiv ; 2023 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-37645747

RESUMO

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted either at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric TMD-targeting compounds as therapeutics, an understanding of the functional and structural basis of their effects on mGluRs is limited. Here we use a battery of approaches to dissect the distinct functional and structural effects of orthosteric versus allosteric ligands. We find using electrophysiological and live cell imaging assays that both agonists and positive allosteric modulators (PAMs) can drive activation and desensitization of mGluRs. The effects of PAMs are pleiotropic, including both the ability to boost the maximal response to orthosteric agonists and to serve independently as desensitization-biased agonists across mGluR subtypes. Conformational sensors reveal PAM-driven inter-subunit re-arrangements at both the LBD and TMD. Motivated by this, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling. Highlights: -Agonists and PAMs drive mGluR activation, desensitization, and endocytosis-PAMs are desensitization-biased and synergistic with agonists-Four combinatorial ligand conditions reveal an ensemble of full-length mGluR structures with novel interfaces-Activation and desensitization involve rolling TMD interfaces which are re-shaped by PAM.

3.
Nat Commun ; 13(1): 3854, 2022 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-35788586

RESUMO

The Kv1.3 potassium channel is expressed abundantly on activated T cells and mediates the cellular immune response. This role has made the channel a target for therapeutic immunomodulation to block its activity and suppress T cell activation. Here, we report structures of human Kv1.3 alone, with a nanobody inhibitor, and with an antibody-toxin fusion blocker. Rather than block the channel directly, four copies of the nanobody bind the tetramer's voltage sensing domains and the pore domain to induce an inactive pore conformation. In contrast, the antibody-toxin fusion docks its toxin domain at the extracellular mouth of the channel to insert a critical lysine into the pore. The lysine stabilizes an active conformation of the pore yet blocks ion permeation. This study visualizes Kv1.3 pore dynamics, defines two distinct mechanisms to suppress Kv1.3 channel activity with exogenous inhibitors, and provides a framework to aid development of emerging T cell immunotherapies.


Assuntos
Canal de Potássio Kv1.3/química , Linfócitos T , Humanos , Imunoglobulinas/metabolismo , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Lisina , Linfócitos T/química
4.
Cell Rep ; 37(4): 109891, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34706237

RESUMO

The kainate receptors (KARs) are members of the ionotropic glutamate receptor family and assemble into tetramers from a pool of five subunit types (GluK1-5). Each subunit confers distinct functional properties to a receptor, but the compositional and stoichiometric diversity of KAR tetramers is not well understood. To address this, we first solve the structure of the GluK1 homomer, which enables a systematic assessment of structural compatibility among KAR subunits. Next, we analyze single-cell RNA sequencing data, which reveal extreme diversity in the combinations of two or more KAR subunits co-expressed within the same cell. We then investigate the composition of individual receptor complexes using single-molecule fluorescence techniques and find that di-heteromers assembled from GluK1, GluK2, or GluK3 can form with all possible stoichiometries, while GluK1/K5, GluK2/K5, and GluK3/K5 can form 3:1 or 2:2 complexes. Finally, using three-color single-molecule imaging, we discover that KARs can form tri- and tetra-heteromers.


Assuntos
Multimerização Proteica , Receptores de Ácido Caínico/química , Receptores de Ácido Caínico/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Células HEK293 , Humanos , Subunidades Proteicas , Receptores de Ácido Caínico/genética
5.
Biochim Biophys Acta ; 1864(12): 1649-1657, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27570147

RESUMO

A periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (CLas-ZnuA2) belong to Cluster A1 family of solute binding proteins (SBPs). The crystal structures in metal-free, intermediate and metal-bound states, in the previous study, revealed the unusual mechanism of metal binding and release for CLas-ZnuA2. Although CLas-ZnuA2 showed maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance seems to be closer to Zn-specific SBPs of Cluster A-I family. The present study reports the binding affinity studies using SPR and CD and crystal structure of CLas-ZnuA2 in Zn2+-bound state. Despite a similar overall structure, there are noticeable differences at the metal binding site. The SPR and CD analysis confirmed our previous observation that CLas-ZnuA2 exhibits a low metal-binding affinity. The low metal-binding affinity of CLas-ZnuA2 could be attributed to the presence of a proline in linker helix resulting in relatively higher bending and rigidity of the same. This structural feature fixes the C-domain similar to metal-bound states of related SBPs. Further, the binding of both Mn2+ and Zn2+ occurs pentavalently with square pyramidal geometry not preferred by either. The site-specific positive Darwinian selection analysis showed that the proline in linker helix is under purifying selection and might have diverged long ago. Our structural and evolutionary analyses suggest that CLasZnua2 might have evolved, particularly for plant pathogens, to facilitate transport of both Mn2+ and Zn2+, with reversible binding to Zn2+, unlike other Mn-binding SBPs (PsaA).


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Fenômenos Biofísicos , Dicroísmo Circular , Cristalografia por Raios X , Genes Bacterianos , Cinética , Modelos Moleculares , Família Multigênica , Filogenia , Conformação Proteica , Rhizobiaceae/genética , Rhizobiaceae/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Zinco/metabolismo
6.
Protein Pept Lett ; 22(8): 750-61, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26021386

RESUMO

A ~56 kDa protein having hemagglutination activity was purified and characterized from the Murraya paniculata seeds. The gel electrophoresis studies demonstrated that protein is primarily of two different subunits, molecular weight ~ 35 and 21 kDa held together by disulfide-linkages and predominantly by secondary forces. The cloning and sequence analysis revealed that the protein exhibited a substantial sequence identity to seed storage 11S globulin family proteins. The sequence analysis of Murraya paniculata globulin (MPG) demonstrated higher and lower molecular weight polypeptides to be acidic (α) and basic (ß) respectively. The sequence analysis further showed that it possesses a characteristic bi-cupin motif and a putative metal binding pocket. CD analysis revealed that the MPG was a ß/α protein with a slightly higher content of the former. Conformational changes in protein have been studied by fluorescence spectrometry by using various chemical treatments. The results demonstrated that MPG belongs to 11S globulin family and exhibit's hemagglutination activity, which implicates it to be possessing lectin-like property.


Assuntos
Murraya/química , Proteínas Recombinantes/química , Proteínas de Armazenamento de Sementes/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Testes de Hemaglutinação , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína
7.
J Struct Biol ; 189(3): 184-94, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25641618

RESUMO

The Znu system, a member of ABC transporter family, is critical for survival and pathogenesis of Candidatus Liberibacter asiaticus (CLA). Two homologues of this system have been identified in CLA. Here, we report high resolution crystal structure of a periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (CLas-ZnuA2) in metal-free, intermediate and metal-bound states. CLas-ZnuA2 showed maximum sequence identity to the Mn/Fe-specific solute binding proteins (SBPs) of cluster A-I family. The overall fold of CLas-ZnuA2 is similar to the related cluster A-I family SBPs. The sequence and structure analysis revealed the unique features of CLas-ZnuA2. The comparison of CLas-ZnuA2 structure in three states showed that metal binding and release is facilitated by a large displacement along with a change in orientation of the side chain for one of the metal binding residue (His39) flipped away from metal binding site in metal-free form. The crystal structure captured in intermediate state of metal binding revealed the changes in conformation and interaction of the loop hosting His39 during the metal binding. A rigid body movement of C-domain along with partial unfolding of linker helix at its C-terminal during metal binding, as reported for PsaA, was not observed in CLas-ZnuA2. The present results suggest that despite showing maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance of CLas-ZnuA2 seems to be closer to Zn-specific SBPs of cluster A-I family.


Assuntos
Proteínas de Bactérias/química , Metais/metabolismo , Proteínas Periplásmicas de Ligação/química , Rhizobiaceae/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Histidina/química , Histidina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Rhizobiaceae/patogenicidade , Homologia de Sequência de Aminoácidos
8.
Appl Biochem Biotechnol ; 174(2): 471-82, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25080380

RESUMO

The present study reports the characterisation of a novel ~12-kDa heterodimeric protein, designated as putrin, from the seeds of Putranjiva roxburghii. The purification of putrin to homogeneity was accomplished using DEAE-sepharose where protein was unbound, CM-sepharose and Cibacron blue 3GA where it was bound and appeared as single peak on a size-exclusion chromatography column. A 15 % sodium dodecyl sulphate polyacrylamide electrophoresis gel, under reducing condition, demonstrated that putrin is made of two polypeptide chains of approximately 4.5 and 7.5 kDa. Circular dichroism studies demonstrated the helical nature and conformational stability of protein at increasing temperatures. Putrin exhibited both RNase and DNase activities and exerted antifungal activity but possessed relatively weak translation-inhibitory activity in cell-free system. The cloning and sequence analysis revealed a 414 bp open reading frame encoding a preproprotein of 137 amino acid residues. The amino acid sequence comparisons and phylogenetic analysis of putrin showed significant homology to 2S seed storage family proteins. The results demonstrated that putrin belongs to 2S albumin family and exhibits a spectrum of biotechnologically exploitable functions.


Assuntos
Albuminas/isolamento & purificação , Antifúngicos/farmacologia , Desoxirribonucleases/metabolismo , Magnoliopsida/química , Ribonucleases/metabolismo , Albuminas/genética , Albuminas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos
9.
Biochem Biophys Res Commun ; 448(4): 349-54, 2014 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-24814706

RESUMO

The plant 2S albumins exhibit a spectrum of biotechnologically exploitable functions. Among them, pumpkin 2S albumin has been shown to possess RNase and cell-free translational inhibitory activities. The present study investigated the anticancer, DNase and antifungal activities of pumpkin 2S albumin. The protein exhibited a strong anticancer activity toward breast cancer (MCF-7), ovarian teratocarcinoma (PA-1), prostate cancer (PC-3 and DU-145) and hepatocellular carcinoma (HepG2) cell lines. Acridine orange staining and DNA fragmentation studies indicated that cytotoxic effect of pumpkin 2S albumin is mediated through induction of apoptosis. Pumpkin 2S albumin showed DNase activity against both supercoiled and linear DNA and exerted antifungal activity against Fusarium oxysporum. Secondary structure analysis by CD showed that protein is highly stable up to 90°C and retains its alpha helical structure. These results demonstrated that pumpkin 2S albumin is a multifunctional protein with host of potential biotechnology applications.


Assuntos
Albuminas 2S de Plantas/química , Albuminas 2S de Plantas/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Cucurbita/química , Desoxirribonucleases/química , Desoxirribonucleases/farmacologia , Apoptose/efeitos dos fármacos , Biotecnologia , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Feminino , Fusarium/efeitos dos fármacos , Células Hep G2 , Humanos , Células MCF-7 , Masculino , Estabilidade Proteica , Estrutura Secundária de Proteína
10.
Proteins ; 82(5): 830-40, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24265134

RESUMO

Murraya koenigii miraculin-like protein (MKMLP) gradually precipitates below pH 7.5. Here, we explore the basis for this aggregation by identifying the aggregation-prone regions via comparative analysis of crystal structures acquired at several pH values. The prediction of aggregation-prone regions showed the presence of four short peptides either in beta sheets or loops on surface of the protein. These peptides were distributed in two patches far apart on the surface. Comparison of crystal structures of MKMLP, determined at 2.2 Å resolution in pH 7.0 and 4.6 in the present study and determined at 2.9 Å in pH 8.0 in an earlier reported study, reveal subtle conformational differences resulting in gradual exposure of aggregation-prone regions. As the pH is lowered, there are alterations in ionic interactions within the protein interactions of the chain with water molecules and exposure of hydrophobic residues. The analysis of symmetry-related molecular interfaces involving one patch revealed shortening of nonpolar intermolecular contacts as the pH decreased. In particular, a decrease in the intermolecular distance between Trp103 of the aggregation-prone peptide WFITTG (103-108) unique to MLPs was observed. These results demonstrated that aggregation occurs due to the cumulative effect of the changes in interactions in two aggregation-prone defined regions.


Assuntos
Murraya/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Aminoácidos/química , Cristalografia por Raios X , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Alinhamento de Sequência
11.
J Mol Evol ; 73(5-6): 369-79, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22274614

RESUMO

Miraculin-like proteins (MLPs) belong to soybean Kunitz super-family and have been characterized from many plant families like Rutaceae, Solanaceae, Rubiaceae, etc. Many of them possess trypsin inhibitory activity and are involved in plant defense. MLPs exhibit significant sequence identity (~30-95%) to native miraculin protein, also belonging to Kunitz super-family compared with a typical Kunitz family member (~30%). The sequence and structure-function comparison of MLPs with that of a classical Kunitz inhibitor have demonstrated that MLPs have evolved to form a distinct group within Kunitz super-family. Sequence analysis of new genes along with available MLP sequences in the literature revealed three major groups for these proteins. A significant feature of Rutaceae MLP type 2 sequences is the presence of phosphorylation motif. Subtle changes are seen in putative reactive loop residues among different MLPs suggesting altered specificities to specific proteases. In phylogenetic analysis, Rutaceae MLP type 1 and type 2 proteins clustered together on separate branches, whereas native miraculin along with other MLPs formed distinct clusters. Site-specific positive Darwinian selection was observed at many sites in both the groups of Rutaceae MLP sequences with most of the residues undergoing positive selection located in loop regions. The results demonstrate the sequence and thereby the structure-function divergence of MLPs as a distinct group within soybean Kunitz super-family due to biotic and abiotic stresses of local environment.


Assuntos
Evolução Molecular , Glycine max/genética , Plantas/genética , Proteínas de Soja/genética , Inibidor da Tripsina de Soja de Kunitz/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Família Multigênica , Filogenia , Seleção Genética , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
12.
Arch Biochem Biophys ; 494(1): 15-22, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19914199

RESUMO

Earlier, the purification of a 21.4kDa protein with trypsin inhibitory activity from seeds of Murraya koenigii has been reported. The present study, based on the amino acid sequence deduced from both cDNA and genomic DNA, establishes it to be a miraculin-like protein and provides crystal structure at 2.9A resolution. The mature protein consists of 190 amino acid residues with seven cysteines arranged in three disulfide bridges. The amino acid sequence showed maximum homology and formed a distinct cluster with miraculin-like proteins, a soybean Kunitz super family member, in phylogenetic analyses. The major differences in sequence were observed at primary and secondary specificity sites in the reactive loop when compared to classical Kunitz family members. The crystal structure analysis showed that the protein is made of twelve antiparallel beta-strands, loops connecting beta-strands and two short helices. Despite similar overall fold, it showed significant differences from classical Kunitz trypsin inhibitors.


Assuntos
Murraya/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cristalografia por Raios X , DNA Complementar , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Homologia de Sequência de Aminoácidos
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