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1.
Blood ; 142(14): 1233-1242, 2023 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-37506345

RESUMO

Human 12-lipoxygenase (12-LOX) is a key enzyme involved in platelet activation, and the regulation of its activity has been targeted for the treatment of heparin-induced thrombocytopenia. Despite the clinical importance of 12-LOX, the exact mechanisms by which it affects platelet activation are not fully understood, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryo-electron microscopy to determine high-resolution structures (1.7-2.8 Å) of human 12-LOX. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may affect its catalytic activity and membrane association. We also identified different conformations within the 12-LOX dimer, which likely represent different time points in its catalytic cycle. Furthermore, we identified small molecules bound to 12-LOX. The active site of the 12-LOX tetramer was occupied by an endogenous 12-LOX inhibitor, a long-chain acyl coenzyme A. In addition, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase 1 clinical trial for the treatment of heparin-induced thrombocytopenia and received a fast-track designation by the Food and Drug Administration. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, their catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.


Assuntos
Ativação Plaquetária , Trombocitopenia , Estados Unidos , Humanos , Microscopia Crioeletrônica , Ácido Araquidônico/metabolismo , Araquidonato 12-Lipoxigenase/metabolismo
2.
J Biol Chem ; 299(7): 104930, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37330172

RESUMO

Psoriasis is a chronic skin disease characterized by hyperproliferative epidermal lesions infiltrated by autoreactive T cells. Individuals expressing the human leukocyte antigen (HLA) C∗06:02 allele are at highest risk for developing psoriasis. An autoreactive T cell clone (termed Vα3S1/Vß13S1) isolated from psoriatic plaques is selective for HLA-C∗06:02, presenting a peptide derived from the melanocyte-specific autoantigen ADAMTSL5 (VRSRRCLRL). Here we determine the crystal structure of this psoriatic TCR-HLA-C∗06:02 ADAMTSL5 complex with a stabilized peptide. Docking of the TCR involves an extensive complementary charge network formed between negatively charged TCR residues interleaving with exposed arginine residues from the self-peptide and the HLA-C∗06:02 α1 helix. We probed these interactions through mutagenesis and activation assays. The charged interface spans the polymorphic region of the C1/C2 HLA group. Notably the peptide-binding groove of HLA-C∗06:02 appears exquisitely suited for presenting highly charged Arg-rich epitopes recognized by this acidic psoriatic TCR. Overall, we provide a structural basis for understanding the engagement of melanocyte antigen-presenting cells by a TCR implicated in psoriasis while simultaneously expanding our knowledge of how TCRs engage HLA-C.


Assuntos
Antígenos HLA-C , Psoríase , Humanos , Eletricidade Estática , Peptídeos/química , Psoríase/patologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas ADAMTS
3.
PLoS Biol ; 19(6): e3001295, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34086670

RESUMO

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores da Colecistocinina/química , Receptores da Colecistocinina/metabolismo , Colecistocinina/metabolismo , Colesterol/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Receptores da Colecistocinina/ultraestrutura , Transdução de Sinais
4.
Purinergic Signal ; 18(4): 421-433, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35821454

RESUMO

Within the family of purinergic receptors, the P2X1 receptor is a ligand-gated ion channel that plays a role in urogenital, immune and cardiovascular function. Specifically, the P2X1 receptor has been implicated in controlling smooth muscle contractions of the vas deferens and therefore has emerged as an exciting drug target for male contraception. In addition, the P2X1 receptor contributes to smooth muscle contractions of the bladder and is a target to treat bladder dysfunction. Finally, platelets and neutrophils have populations of P2X1 receptors that could be targeted for thrombosis and inflammatory conditions. Drugs that specifically target the P2X1 receptor have been challenging to develop, and only recently have small molecule antagonists of the P2X1 receptor been available. However, these ligands need further biological validation for appropriate selectivity and drug-like properties before they will be suitable for use in preclinical models of disease. Although the atomic structure of the P2X1 receptor has yet to be determined, the recent discovery of several other P2X receptor structures and improvements in the field of structural biology suggests that this is now a distinct possibility. Such efforts may significantly improve drug discovery efforts at the P2X1 receptor.


Assuntos
Receptores Purinérgicos P2X1 , Masculino , Humanos , Bexiga Urinária , Contração Muscular , Ducto Deferente/fisiologia , Plaquetas , Receptores Purinérgicos P2X , Trifosfato de Adenosina
5.
J Biol Chem ; 292(42): 17203-17215, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28855257

RESUMO

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.


Assuntos
Proteínas ADAMTS , Apresentação de Antígeno , Antígenos HLA-C , Peptídeos , Proteínas ADAMTS/química , Proteínas ADAMTS/genética , Proteínas ADAMTS/imunologia , Proteínas ADAMTS/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Antígenos HLA-C/química , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Antígenos HLA-C/metabolismo , Humanos , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Peptídeos/metabolismo
6.
Chembiochem ; 19(3): 229-238, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29193585

RESUMO

The ß subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (ß1 and ß2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the ß1- and ß2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. ß2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into ß2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why ß2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for ß2 and Thr for ß1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant ß2-Thr101Δ/Val134Thr differs from that of ß1-CBM, and mutant ß1-Thr101ins/Thr134Val differs from that of ß2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type ß-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant ß1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of ß2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Carboidratos/química , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Sítios de Ligação , Calorimetria , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Ratos , Termodinâmica
7.
Biochem J ; 468(2): 245-57, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25774984

RESUMO

AMP-activated protein kinase (AMPK) is an αßγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The ß-subunit exists in two isoforms (ß1 and ß2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (ß1- and ß2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. ß2-CBM binds linear carbohydrates with 4-fold greater affinity than ß1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of ß2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-ß-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of ß1- and ß2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of ß2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of ß2-CBM. In contrast, the α1,6 branch could not be observed in the ß1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to ß1- and ß2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific ß2-isoform may have greater affinity for partially degraded glycogen.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Glicogênio/metabolismo , Oligossacarídeos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Isoenzimas , Cinética , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Especificidade por Substrato
8.
Curr Opin Struct Biol ; 79: 102560, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36848776

RESUMO

G-protein coupled receptors (GPCRs) are important therapeutic targets for the treatment of human disease. Although GPCRs are highly successful drug targets, there are many challenges associated with the discovery and translation of small molecule ligands that target the endogenous ligand-binding site for GPCRs. Allosteric modulators are a class of ligands that target alternative binding sites known as allosteric sites and offer fresh opportunities for the development of new therapeutics. However, only a few allosteric modulators have been approved as drugs. Advances in GPCR structural biology enabled by the cryogenic electron microscopy (cryo-EM) revolution have provided new insights into the molecular mechanism and binding location of small molecule allosteric modulators. This review highlights the latest findings from allosteric modulator-bound structures of Class A, B, and C GPCRs with a focus on small molecule ligands. Emerging methods that will facilitate cryo-EM structures of more difficult ligand-bound GPCR complexes are also discussed. The results of these studies are anticipated to aid future structure-based drug discovery efforts across many different GPCRs.


Assuntos
Regulação Alostérica , Microscopia Crioeletrônica , Receptores Acoplados a Proteínas G , Animais , Humanos , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Conformação Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestrutura
9.
Nat Commun ; 14(1): 5440, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37673901

RESUMO

The M4 muscarinic acetylcholine receptor (M4 mAChR) has emerged as a drug target of high therapeutic interest due to its expression in regions of the brain involved in the regulation of psychosis, cognition, and addiction. The mAChR agonist, xanomeline, has provided significant improvement in the Positive and Negative Symptom Scale (PANSS) scores in a Phase II clinical trial for the treatment of patients suffering from schizophrenia. Here we report the active state cryo-EM structure of xanomeline bound to the human M4 mAChR in complex with the heterotrimeric Gi1 transducer protein. Unexpectedly, two molecules of xanomeline were found to concomitantly bind to the monomeric M4 mAChR, with one molecule bound in the orthosteric (acetylcholine-binding) site and a second molecule in an extracellular vestibular allosteric site. Molecular dynamic simulations supports the structural findings, and pharmacological validation confirmed that xanomeline acts as a dual orthosteric and allosteric ligand at the human M4 mAChR. These findings provide a basis for further understanding xanomeline's complex pharmacology and highlight the myriad of ways through which clinically relevant ligands can bind to and regulate GPCRs.


Assuntos
Comportamento Aditivo , Humanos , Sítio Alostérico , Encéfalo , Cognição
10.
Elife ; 122023 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-37248726

RESUMO

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.


Assuntos
Receptor Muscarínico M4 , Receptores Muscarínicos , Humanos , Acetilcolina/metabolismo , Regulação Alostérica , Sítio Alostérico , Microscopia Crioeletrônica , Ligantes , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/metabolismo
11.
Biophys J ; 102(2): 305-14, 2012 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-22339867

RESUMO

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the ß-subunit, for which there are two isoforms (ß(1) and ß(2)). Muscle-specific ß(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous ß(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of ß(2)-CBM is concurrent with an increase in flexibility in ß(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to ß(1)-CBM, unbound ß(2)-CBM showed microsecond-to-millisecond motion at the base of a ß-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this ß-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from ß(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the ß(2)-CBM mutant. Insertion of a threonine into the background of ß(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Metabolismo dos Carboidratos , Modelos Moleculares , Movimento , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Sequência de Aminoácidos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato
12.
Methods Mol Biol ; 1732: 87-98, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29480470

RESUMO

Understanding the kinetics of proteins interacting with their ligands is important for characterizing molecular mechanism. However, it can be difficult to determine the extent and nature of these interactions for weakly formed protein-ligand complexes that have lifetimes of micro- to milliseconds. Nuclear magnetic resonance (NMR) spectroscopy is a powerful solution-based method for the atomic-level analysis of molecular interactions on a wide range of timescales, including micro- to milliseconds. Recently the combination of thermodynamic experiments using isothermal titration calorimetry (ITC) with kinetic measurements using ZZ-exchange and CPMG relaxation dispersion NMR spectroscopy have been used to determine the kinetics of weakly interacting protein systems. This chapter describes the application of ITC and NMR to understand the differences in the kinetics of carbohydrate binding by the ß1- and ß2-carbohydrate-binding modules of AMP-activated protein kinase.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Calorimetria/métodos , Carboidratos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Calorimetria/instrumentação , Cinética , Ligantes , Ressonância Magnética Nuclear Biomolecular/instrumentação , Ligação Proteica , Termodinâmica
13.
Nat Commun ; 8: 15924, 2017 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-28649982

RESUMO

Expression of HLA-C varies widely across individuals in an allele-specific manner. This variation in expression can influence efficacy of the immune response, as shown for infectious and autoimmune diseases. MicroRNA binding partially influences differential HLA-C expression, but the additional contributing factors have remained undetermined. Here we use functional and structural analyses to demonstrate that HLA-C expression is modulated not just at the RNA level, but also at the protein level. Specifically, we show that variation in exons 2 and 3, which encode the α1/α2 domains, drives differential expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors that modulate HLA-C expression.


Assuntos
Antígenos HLA-C/química , Antígenos HLA-C/genética , Alelos , Animais , Éxons , Regulação da Expressão Gênica , Variação Genética , Antígenos HLA-C/metabolismo , Humanos , Mamíferos/classificação , Mamíferos/genética , Pan troglodytes , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Filogenia , Regiões Promotoras Genéticas , Ligação Proteica
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