RESUMO
Mutation in leucine-rich repeat (LRR) kinase 2 (LRRK2) is a common cause of Parkinson's disease (PD). Aberrant LRRK2 kinase activity is associated with disease pathogenesis and thus it is an attractive drug target for combating PD. Intense efforts in the past nearly two decades have focused on the development of small-molecule inhibitors of the kinase domain of LRRK2 and have identified potent kinase inhibitors. However, most LRRK2 kinase inhibitors have shown adverse effects; therefore, alternative-mechanism-based strategies are desperately needed. In this review, we discuss the new insights gleaned from recent cryoelectron microscope (cryo-EM) structures of LRRK2 towards understanding the mechanisms of actions of LRRK2 and explore the potential new therapeutic avenues.
Assuntos
Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases , MutaçãoRESUMO
GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.
Assuntos
Sítio Alostérico , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
Mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mutação de Sentido Incorreto , Doença de Parkinson , Multimerização Proteica , Substituição de Aminoácidos , Guanosina Difosfato/química , Guanosina Difosfato/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Domínios ProteicosRESUMO
Parkinson disease-associated mutations within the GTPase domain Ras of complex proteins (ROC) of leucine rich repeat kinase 2 (LRRK2) result in an abnormal over-activation of its kinase domain. However, the mechanisms involved remain unclear. Recent study has shown that LRRK2 G-domain cycles between monomeric and dimeric conformations upon binding to GTP or guanosine diphosphate, and that the Parkinson's disease (PD)-associated R1441C/G/H mutations impair the G-domain monomer-dimer dynamics and trap the G-domain in a constitutive monomeric conformation. That led us to question whether other disease-associated mutations in G-domain would also affect its conformation. Here, we report that another PD-associated N1437H mutation also impairs its monomer-dimer conformational dynamics and GTPase activity. In contrast with mutations at R1441, ROCN1437H was found to be locked in a stable dimeric conformation in solution and its GTPase activity was â¼4-fold lower than that of the wild-type. Furthermore, the N1437H mutation reduced the GTP binding affinity by â¼2.5-fold when compared with other pathogenic G-domain mutations. Moreover, ROCN1437H was found to have a slower GTP dissociation rate, indicating that N1437H might interrupt the nucleotide exchange cycle. Taken together, our data support that conformational dynamics is important for LRRK2 GTPase activity and that the N1437H mutation impairs GTPase activity by locking the ROC domain in a persistently dimeric state.-Huang, X., Wu, C., Park, Y., Long, X., Hoang, Q. Q., Liao, J. The Parkinson's disease-associated mutation N1437H impairs conformational dynamics in the G domain of LRRK2.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/genética , Western Blotting , Cromatografia em Gel , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética , Conformação ProteicaRESUMO
In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.
Assuntos
Antibacterianos/farmacologia , Chaperonina 60/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Nitrofuranos/farmacologia , Pró-Fármacos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Chaperonina 60/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nitrofuranos/síntese química , Nitrofuranos/química , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-AtividadeRESUMO
All living organisms contain a unique class of molecular chaperones called 60â¯kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.
Assuntos
Produtos Biológicos/química , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Rafoxanida/química , Salicilanilidas/química , Suramina/química , Produtos Biológicos/metabolismo , Chaperonina 10/antagonistas & inibidores , Chaperonina 60/antagonistas & inibidores , Escherichia coli/metabolismo , Humanos , Concentração Inibidora 50 , Dobramento de Proteína , Rafoxanida/metabolismo , Salicilanilidas/metabolismo , Suramina/metabolismoRESUMO
Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.
Assuntos
Chaperonina 60/uso terapêutico , Mycobacterium tuberculosis/patogenicidade , Tuberculose/tratamento farmacológico , Proteínas de Bactérias/metabolismo , Chaperonina 60/farmacologia , Humanos , Modelos Moleculares , PolifarmacologiaRESUMO
The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.
Assuntos
Cromatina/química , DNA/química , Genoma , Proteínas com Domínio T/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cromatina/metabolismo , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Elementos Facilitadores Genéticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Sequências Repetidas Invertidas , Camundongos , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Xenopus laevisRESUMO
Multiple system atrophy (MSA) is a sporadic orphan neurodegenerative disorder. No treatment is currently available to slow down the aggressive neurodegenerative process, and patients die within a few years after disease onset. The cytopathological hallmark of MSA is the accumulation of alpha-synuclein (α-syn) aggregates in affected oligodendrocytes. Several studies point to α-syn oligomerization and aggregation as a mediator of neurotoxicity in synucleinopathies including MSA. C-terminal truncation by the inflammatory protease caspase-1 has recently been implicated in the mechanisms that promote aggregation of α-syn in vitro and in neuronal cell models of α-syn toxicity. We present here an in vivo proof of concept of the ability of the caspase-1 inhibitor prodrug VX-765 to mitigate α-syn pathology and to mediate neuroprotection in proteolipid protein α-syn (PLP-SYN) mice, a transgenic mouse model of MSA. PLP-SYN and age-matched wild-type mice were treated for a period of 11 wk with VX-765 or placebo. VX-765 prevented motor deficits in PLP-SYN mice compared with placebo controls. More importantly, VX-765 was able to limit the progressive toxicity of α-syn aggregation by reducing its load in the striatum of PLP-SYN mice. Not only did VX-765 reduce truncated α-syn, but it also decreased its monomeric and oligomeric forms. Finally, VX-765 showed neuroprotective effects by preserving tyrosine hydroxylase-positive neurons in the substantia nigra of PLP-SYN mice. In conclusion, our results suggest that VX-765, a drug that was well tolerated in a 6 wk-long phase II trial in patients with epilepsy, is a promising candidate to achieve disease modification in synucleinopathies by limiting α-syn accumulation.
Assuntos
Caspase 1/genética , Corpo Estriado/efeitos dos fármacos , Dipeptídeos/farmacologia , Atrofia de Múltiplos Sistemas/tratamento farmacológico , Oligodendroglia/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , alfa-Sinucleína/genética , para-Aminobenzoatos/farmacologia , Animais , Caspase 1/metabolismo , Ensaios Clínicos como Assunto , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/genética , Proteólise , Transdução de Sinais , Substância Negra/metabolismo , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.
Assuntos
Caspase 1/genética , Inflamassomos/metabolismo , Corpos de Lewy/metabolismo , Neurônios/metabolismo , Agregados Proteicos/genética , alfa-Sinucleína/genética , Compostos de Alúmen/farmacologia , Caspase 1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Corpos de Lewy/efeitos dos fármacos , Corpos de Lewy/patologia , Lipopolissacarídeos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Nigericina/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Vitamina K 3/farmacologia , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , para-Aminobenzoatos/farmacologiaRESUMO
Small G-proteins are structurally-conserved modules that function as molecular on-off switches. They function in many different cellular processes with differential specificity determined by the unique effector-binding surfaces, which undergo conformational changes during the switching action. These switches are typically standalone monomeric modules that form transient heterodimers with specific effector proteins in the 'on' state, and cycle to back to the monomeric conformation in the 'off' state. A new class of small G-proteins called "Roco" was discovered about a decade ago; this class is distinct from the typical G-proteins in several intriguing ways. Their switch module resides within a polypeptide chain of a large multi-domain protein, always adjacent to a unique domain called COR, and its effector kinase often resides within the same polypeptide. As such, the mechanisms of action of the Roco G-proteins are likely to differ from those of the typical G-proteins. Understanding these mechanisms is important because aberrant activity in the human Roco protein LRRK2 is associated with the pathogenesis of Parkinson's disease. This review provides an update on the current state of our understanding of the Roco G-proteins and the prospects of targeting them for therapeutic purposes.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Doença de Parkinson/patologiaRESUMO
Pharmacological chaperones are small molecules that bind to proteins and stabilize them against thermal denaturation or proteolytic degradation, as well as assist or prevent certain protein-protein assemblies. These activities are being exploited for the development of treatments for diseases caused by protein instability and/or aberrant protein-protein interactions, such as those found in certain forms of cancers and neurodegenerative diseases. However, designing or discovering pharmacological chaperones for specific targets is challenging because of the relatively featureless protein target surfaces, the lack of suitable chemical libraries, and the shortage of efficient high-throughput screening methods. In this study, we attempted to address all these challenges by synthesizing a diverse library of small molecules that mimic protein α-helical secondary structures commonly found in protein-protein interaction surfaces. This was accompanied by establishing a facile "on-bead" high-throughput screening method that allows for rapid and efficient discovery of potential pharmacological chaperones and for identifying novel chaperones/inhibitors against a cancer-associated protein, myeloid cell leukemia 1 (MCL-1), and a Parkinson disease-associated protein, α-synuclein. Our data suggest that the compounds and methods described here will be useful tools for the development of pharmaceuticals for complex-disease targets that are traditionally deemed "undruggable."
Assuntos
Descoberta de Drogas , Chaperonas Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neoplasias , Doença de Parkinson , alfa-Sinucleína , Humanos , Células Jurkat , Chaperonas Moleculares/síntese química , Chaperonas Moleculares/química , Chaperonas Moleculares/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/metabolismoRESUMO
Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (RocR1441H) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Doença de Parkinson/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/genética , Western Blotting , Cromatografia em Gel , Dicroísmo Circular , Dimerização , Eletroforese em Gel de Poliacrilamida , GTP Fosfo-Hidrolases/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Espectrometria de Massas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Bacillus subtilis GabR is a transcription factor that regulates gamma-aminobutyric acid (GABA) metabolism. GabR is a member of the understudied MocR/GabR subfamily of the GntR family of transcription regulators. A typical MocR/GabR-type regulator is a chimeric protein containing a short N-terminal helix-turn-helix DNA-binding domain and a long C-terminal pyridoxal 5'-phosphate (PLP)-binding putative aminotransferase domain. In the presence of PLP and GABA, GabR activates the gabTD operon, which allows the bacterium to use GABA as nitrogen and carbon sources. GabR binds to its own promoter and represses gabR transcription in the absence of GABA. Here, we report two crystal structures of full-length GabR from B. subtilis: a 2.7-Å structure of GabR with PLP bound and the 2.55-Å apo structure of GabR without PLP. The quaternary structure of GabR is a head-to-tail domain-swap homodimer. Each monomer comprises two domains: an N-terminal winged-helix DNA-binding domain and a C-terminal PLP-binding type I aminotransferase-like domain. The winged-helix domain contains putative DNA-binding residues conserved in other GntR-type regulators. Together with sedimentation velocity and fluorescence polarization assays, the crystal structure of GabR provides insights into DNA binding by GabR at the gabR and gabT promoters. The absence of GabR-mediated aminotransferase activity in the presence of GABA and PLP, and the presence of an active site configuration that is incompatible with stabilization of the GABA external aldimine suggest that a GabR aminotransferase-like activity involving GABA and PLP is not essential to its primary function as a transcription regulator.
Assuntos
Bacillus subtilis/química , Evolução Molecular , Regulação Bacteriana da Expressão Gênica/genética , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/química , Dimerização , Fosfato de Piridoxal/metabolismo , Fatores de Transcrição/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 10(6) M(-1) s(-1) for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 µM for preferred substrates) compared to those of typical AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" α-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed.
Assuntos
Hidrolases de Éster Carboxílico/química , Chryseobacterium/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Chryseobacterium/química , Chryseobacterium/fisiologia , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Percepção de Quorum , Especificidade por SubstratoRESUMO
Mutations of DJ-1 cause familial Parkinson's disease (PD), although the role of DJ-1 in PD remains unresolved. Very recent reports have shown that DJ-1 interacts with copper ions. This evidence opens new avenues to understanding the function of DJ-1 and its role in PD. Herein, we report that Zn(II) binds to DJ-1 with great selectivity among the other metals examined: Mn(II), Fe(II), Co(II), Ni(II), and Cu(II). High-resolution X-ray crystallography (1.18 Å resolution) shows Zn(II) is coordinated to the protein by the key residues Cys106 and Glu18. These results suggest that DJ-1 may be regulated and/or stabilized by Zn(II).
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Zinco/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Proteínas Oncogênicas/química , Conformação Proteica , Proteína Desglicase DJ-1 , TermodinâmicaRESUMO
A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein.
Assuntos
Modelos Moleculares , Polímeros/química , Estrutura Secundária de Proteína , alfa-Sinucleína/química , Dicroísmo Circular , Humanos , Microscopia Eletrônica , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Ribosomal protein L11 (RPL11) has been shown to activate p53 by binding to MDM2 and negating its p53 suppression activity in response to ribosomal stress. Although a mutation at Cys-305 within the zinc finger domain of MDM2 has been shown to drastically impair MDM2 interaction with RPL11 and thus escapes the inhibition by this ribosomal protein, it still remains elusive whether RPL11 inactivates MDM2 via direct action on this zinc finger domain and what is the chemical nature of this specific interaction. To define the roles of the MDM2 zinc finger in association with RPL11, we conducted hydrogen-deuterium exchange mass spectrometry, computational modeling, circular dichroism, and mutational analyses of the zinc finger domain of MDM2 and human RPL11. Our study reveals that RPL11 forms a stable complex with MDM2 in vitro through direct contact with its zinc finger. This binding is disrupted by single mutations of non-cysteine amino acids within the zinc finger domain of MDM2. Basic residues in RPL11 are crucial for the stable binding and RPL11 suppression of MDM2 activity toward p53. These results provide the first line of evidence for the specific interaction between RPL11 and the zinc finger of MDM2 via hydrophilic residues as well as a molecular foundation for better understanding RPL11 inhibition of MDM2 function.