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1.
Nat Commun ; 12(1): 5885, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620873

RESUMO

Pathogenic fungi exhibit a heavy burden on medical care and new therapies are needed. Here, we develop the fungal specific enzyme sterylglucosidase 1 (Sgl1) as a therapeutic target. Sgl1 converts the immunomodulatory glycolipid ergosterol 3ß-D-glucoside to ergosterol and glucose. Previously, we found that genetic deletion of Sgl1 in the pathogenic fungus Cryptococcus neoformans (Cn) results in ergosterol 3ß-D-glucoside accumulation, renders Cn non-pathogenic, and immunizes mice against secondary infections by wild-type Cn, even in condition of CD4+ T cell deficiency. Here, we disclose two distinct chemical classes that inhibit Sgl1 function in vitro and in Cn cells. Pharmacological inhibition of Sgl1 phenocopies a growth defect of the Cn Δsgl1 mutant and prevents dissemination of wild-type Cn to the brain in a mouse model of infection. Crystal structures of Sgl1 alone and with inhibitors explain Sgl1's substrate specificity and enable the rational design of antifungal agents targeting Sgl1.

2.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
3.
Adv Biol Regul ; 79: 100783, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33495125

RESUMO

Mammalian phospholipase D (PLD) generates phosphatidic acid, a dynamic lipid secondary messenger involved with a broad spectrum of cellular functions including but not limited to metabolism, migration, and exocytosis. As a promising pharmaceutical target, the biochemical properties of PLD have been well characterized. This has led to the recent crystal structures of human PLD1 and PLD2, the development of PLD specific pharmacological inhibitors, and the identification of cellular regulators of PLD. In this review, we discuss the PLD1 and PLD2 structures, PLD inhibition by small molecules, and the regulation of PLD activity by effector proteins and lipids.

4.
SLAS Discov ; 26(1): 113-121, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32734807

RESUMO

There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC50 and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).

5.
Nat Commun ; 11(1): 1734, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32242008

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

6.
Nat Chem Biol ; 16(4): 400-407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32198492

RESUMO

The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we present a 1.8 Å-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target.


Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Fosfolipase D/ultraestrutura , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células COS , Catálise , Domínio Catalítico , Chlorocebus aethiops , Humanos , Lipídeos de Membrana , Fosfatidilcolinas , Ligação Proteica , Proteína Quinase C/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais/efeitos dos fármacos
7.
Nat Commun ; 11(1): 1309, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161260

RESUMO

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.


Assuntos
Fosfatidato Fosfatase/metabolismo , Fosfatidato Fosfatase/ultraestrutura , Proteínas de Protozoários/ultraestrutura , Domínio Catalítico/genética , Cristalografia por Raios X , Células HEK293 , Humanos , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/isolamento & purificação , Conformação Proteica em alfa-Hélice , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura , Tetrahymena thermophila/enzimologia , Transfecção
8.
J Biol Chem ; 294(18): 7488-7502, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30890560

RESUMO

Neutral sphingomyelinase 2 (nSMase2) produces the bioactive lipid ceramide and has important roles in neurodegeneration, cancer, and exosome formation. Although nSMase2 has low basal activity, it is fully activated by phosphatidylserine (PS). Previous work showed that interdomain interactions within nSMase2 are needed for PS activation. Here, we use multiple approaches, including small angle X-ray scattering, hydrogen-deuterium exchange-MS, circular dichroism and thermal shift assays, and membrane yeast two-hybrid assays, to define the mechanism mediating this interdomain interactions within nSMase2. In contrast to what we previously assumed, we demonstrate that PS binding at the N-terminal and juxtamembrane regions of nSMase2 rather acts as a conformational switch leading to interdomain interactions that are critical to enzyme activation. Our work assigns a unique function for a class of linkers of lipid-activated, membrane-associated proteins. It indicates that the linker actively participates in the activation mechanism via intramolecular interactions, unlike the canonical linkers that typically aid protein dimerization or localization.


Assuntos
Esfingomielina Fosfodiesterase/metabolismo , Regulação Alostérica , Aminoácidos/química , Domínio Catalítico , Ativação Enzimática , Humanos , Hidroxiureia/farmacologia , Mutação , Conformação Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Esfingomielina Fosfodiesterase/química , Esfingomielina Fosfodiesterase/genética , Difração de Raios X
9.
J Lipid Res ; 59(1): 162-170, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29092960

RESUMO

Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast Saccharomyces cerevisiae An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin Ki for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine α-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH3+ base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.


Assuntos
Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Serina C-Palmitoiltransferase/metabolismo , Esfingosina/análogos & derivados , Cromatografia Líquida de Alta Pressão , Ácidos Graxos Monoinsaturados/farmacologia , Serina C-Palmitoiltransferase/antagonistas & inibidores , Espectrometria de Massas por Ionização por Electrospray , Esfingosina/análise , Esfingosina/metabolismo , Espectrometria de Massas em Tandem
10.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1862(10 Pt B): 1131-1145, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28642195

RESUMO

Triacylglycerols (TAG) serve as the predominant form of energy storage in mammalian cells, and TAG synthesis influences conditions such as obesity, fatty liver, and insulin resistance. In most tissues, the glycerol 3-phosphate pathway enzymes are responsible for TAG synthesis, and the regulation and function of these enzymes is therefore important for metabolic homeostasis. Here we review the sites and regulation of glycerol-3-phosphate acyltransferase (GPAT), acylglycerol-3-phosphate acyltransferase (AGPAT), lipin phosphatidic acid phosphatase (PAP), and diacylglycerol acyltransferase (DGAT) enzyme action. We highlight the critical roles that these enzymes play in human health by reviewing Mendelian disorders that result from mutation in the corresponding genes. We also summarize the valuable insights that genetically engineered mouse models have provided into the cellular and physiological roles of GPATs, AGPATs, lipins and DGATs. Finally, we comment on the status and feasibility of therapeutic approaches to metabolic disease that target enzymes of the glycerol 3-phosphate pathway. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase , Glicerol-3-Fosfato O-Aciltransferase , Gotículas Lipídicas/metabolismo , Fosfatidato Fosfatase , Triglicerídeos , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Animais , Glicerol-3-Fosfato O-Aciltransferase/genética , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Humanos , Camundongos , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Triglicerídeos/genética , Triglicerídeos/metabolismo
11.
Proc Natl Acad Sci U S A ; 114(28): E5549-E5558, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28652336

RESUMO

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.


Assuntos
Sítio Alostérico , Ceramidas/biossíntese , Esfingomielina Fosfodiesterase/química , Compostos de Anilina/química , Compostos de Benzilideno/química , Domínio Catalítico , Membrana Celular/metabolismo , Cristalografia por Raios X , Humanos , Lipídeos/química , Células MCF-7 , Ligação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae , Transdução de Sinais
12.
Structure ; 23(8): 1482-1491, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26190575

RESUMO

Neutral ceramidase (nCDase) catalyzes conversion of the apoptosis-associated lipid ceramide to sphingosine, the precursor for the proliferative factor sphingosine-1-phosphate. As an enzyme regulating the balance of ceramide and sphingosine-1-phosphate, nCDase is emerging as a therapeutic target for cancer. Here, we present the 2.6-Å crystal structure of human nCDase in complex with phosphate that reveals a striking, 20-Å deep, hydrophobic active site pocket stabilized by a eukaryotic-specific subdomain not present in bacterial ceramidases. Utilizing flexible ligand docking, we predict a likely binding mode for ceramide that superimposes closely with the crystallographically observed transition state analog phosphate. Our results suggest that nCDase uses a new catalytic strategy for Zn(2+)-dependent amidases, and generates ceramide specificity by sterically excluding sphingolipids with bulky headgroups and specifically recognizing the small hydroxyl head group of ceramide. Together, these data provide a foundation to aid drug development and establish common themes for how proteins recognize the bioactive lipid ceramide.


Assuntos
Ceramidas/química , Lisofosfolipídeos/química , Ceramidase Neutra/química , Esfingosina/análogos & derivados , Esfingosina/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Ceramidas/metabolismo , Cristalografia por Raios X , Escherichia coli/química , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Lisofosfolipídeos/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ceramidase Neutra/genética , Ceramidase Neutra/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Células Sf9 , Especificidade da Espécie , Esfingosina/metabolismo , Spodoptera
13.
Adv Biol Regul ; 57: 24-41, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25465297

RESUMO

Our understanding of the functions of ceramide signaling has advanced tremendously over the past decade. In this review, we focus on the roles and regulation of neutral sphingomyelinase 2 (nSMase2), an enzyme that generates the bioactive lipid ceramide through the hydrolysis of the membrane lipid sphingomyelin. A large body of work has now implicated nSMase2 in a diverse set of cellular functions, physiological processes, and disease pathologies. We discuss different aspects of this enzyme's regulation from transcriptional, post-translational, and biochemical. Furthermore, we highlight nSMase2 involvement in cellular processes including inflammatory signaling, exosome generation, cell growth, and apoptosis, which in turn play important roles in pathologies such as cancer metastasis, Alzheimer's disease, and other organ systems disorders. Lastly, we examine avenues where targeted nSMase2-inhibition may be clinically beneficial in disease scenarios.


Assuntos
Doença de Alzheimer/metabolismo , Apoptose , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Doença de Alzheimer/genética , Animais , Ceramidas/genética , Ceramidas/metabolismo , Exossomos/genética , Exossomos/metabolismo , Humanos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Processamento de Proteína Pós-Traducional/genética , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/genética , Esfingomielinas/metabolismo , Transcrição Genética/genética
14.
PLoS One ; 9(8): e105830, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25144372

RESUMO

Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases.


Assuntos
Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Ralstonia solanacearum/enzimologia , Esfingomielina Fosfodiesterase/química , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Hidrólise , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/química , Esfingomielinas/metabolismo , Especificidade por Substrato
15.
Handb Exp Pharmacol ; (215): 57-76, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23579449

RESUMO

Sphingolipids are an important class of lipid molecules that play fundamental roles in our cells and body. Beyond a structural role, it is now clearly established that sphingolipids serve as bioactive signaling molecules to regulate diverse processes including inflammatory signaling, cell death, proliferation, and pain sensing. Sphingolipid metabolites have been implicated in the onset and progression of various diseases including cancer, lung disease, diabetes, and lysosomal storage disorders. Here we review sphingolipid metabolism to introduce basic concepts as well as emerging complexities in sphingolipid function gained from modern technological advances and detailed cell and animal studies. Furthermore, we discuss the family of neutral sphingomyelinases (N-SMases), which generate ceramide through the hydrolysis of sphingomyelin and are key enzymes in sphingolipid metabolism. Four mammalian N-SMase enzymes have now been identified. Most prominent is nSMase2 with established roles in bone mineralization, exosome formation, and cellular stress responses. Function for the other N-SMases has been more enigmatic and is an area of active investigation. The known properties and potential role(s) of each enzyme are discussed to help guide future studies.


Assuntos
Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Animais , Desenvolvimento Ósseo , Humanos , Transdução de Sinais , Esfingolipídeos/química
16.
PLoS Biol ; 11(2): e1001479, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23424282

RESUMO

HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between "on" and "off" conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.


Assuntos
Proteínas de Bactérias/química , Células Quimiorreceptoras/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais
17.
J Mol Biol ; 425(5): 886-901, 2013 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-23274111

RESUMO

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per-Arnt-Sim) and poly-HAMP (histidine kinase-adenylyl cyclase-methyl-accepting chemotaxis protein-phosphatase) domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS but propagate to the Iß strand in a manner predicted to alter PAS-PAS associations and the downstream HAMP junction within full-length Aer2. Small-angle X-ray scattering of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initio small-angle X-ray scattering molecular envelopes, and pulsed dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the Escherichia coli Aer paradigm, where side-on PAS-HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Heme/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intercelular , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Sistemas de Secreção Tipo III
19.
Structure ; 18(4): 436-48, 2010 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-20399181

RESUMO

HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.


Assuntos
Estrutura Terciária de Proteína , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Biologia Computacional/métodos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas/química , Selenometionina/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Solventes/química
20.
Biochemistry ; 49(20): 4327-38, 2010 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-20411915

RESUMO

The mammalian circadian clock synchronizes physical and metabolic activity with the diurnal cycle through a transcriptional-posttranslational feedback loop. An additional feedback mechanism regulating clock timing has been proposed to involve oscillation in heme availability. Period 2 (PER2), an integral component in the negative feedback loop that establishes circadian rhythms in mammals, has been identified as a heme-binding protein. However, the majority of evidence for heme binding is based upon in vitro heme-binding assays. We sought to ascertain if these largely spectral assays could distinguish between specific and nonspecific heme interactions. Heme-binding properties by a number of other well-characterized proteins, all with no known biological role involving heme interaction, corresponded to those displayed by PER2. Site-directed mutants of putative heme-binding residues identified by MCD were unable to locate a specific heme-binding site on PER2. Protein film electrochemistry also indicates that heme binds PER2 nonspecifically on the protein surface. Our results establish the inability of qualitative in vitro assays to easily distinguish between specific and nonspecific heme binding. We conclude that heme binding to PER2 is likely to be nonspecific and does not involve the hydrophobic pocket within the PER2 PAS domains that in other PAS proteins commonly recognizes cofactors. These findings also question the significance of in vivo studies that implicate heme interactions with the clock proteins PER2 and nPAS2 in biological function.


Assuntos
Heme/metabolismo , Proteínas Circadianas Period/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Dicroísmo Circular , Heme/química , Humanos , Ferro/metabolismo , Mamíferos/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Proteínas Circadianas Period/química , Proteínas Circadianas Period/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Multimerização Proteica/genética , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
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