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2.
Nat Struct Mol Biol ; 28(4): 388-397, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33782614

RESUMO

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.


Assuntos
Proteínas AAA/genética , Katanina/genética , Proteínas Associadas aos Microtúbulos/genética , Piridinas/química , Proteínas AAA/antagonistas & inibidores , Proteínas AAA/ultraestrutura , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Humanos , Katanina/ultraestrutura , Proteínas Associadas aos Microtúbulos/ultraestrutura , Microtúbulos/genética , Microtúbulos/ultraestrutura , Conformação Proteica/efeitos dos fármacos , Domínios Proteicos/genética , Piridinas/farmacologia , Triazóis/química
3.
Elife ; 92020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33295875

RESUMO

This article is dedicated to the memory of Michael G. Rossmann. Dating back to the last universal common ancestor, P-loop NTPases and Rossmanns comprise the most ubiquitous and diverse enzyme lineages. Despite similarities in their overall architecture and phosphate binding motif, a lack of sequence identity and some fundamental structural differences currently designates them as independent emergences. We systematically searched for structure and sequence elements shared by both lineages. We detected homologous segments that span the first ßαß motif of both lineages, including the phosphate binding loop and a conserved aspartate at the tip of ß2. The latter ligates the catalytic metal in P-loop NTPases, while in Rossmanns it binds the nucleotide's ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the ß2-Asp to take either one of these two roles. While convergence cannot be completely ruled out, we show that both lineages likely emerged from a common ßαß segment that comprises the core of these enzyme families to this very day.


Assuntos
Proteínas AAA/metabolismo , Proteínas AAA/química , Proteínas AAA/genética , Sítios de Ligação , Evolução Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência
4.
J Mol Biol ; 432(20): 5544-5564, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32750390

RESUMO

A hallmark of the catalytically essential Walker B motif of P-loop NTPases is the presence of an acidic residue (aspartate/glutamate) for efficient Mg2+ coordination. Although the Walker B motif has been identified in well-studied examples of P-loop NTPases, its identity is ambiguous in many families, for example, in the prokaryotic small Ras-like GTPase family of MglA. MglA, belonging to TRAFAC class of P-loop NTPases, possesses a threonine at the position equivalent to Walker B aspartate in eukaryotic Ras-like GTPases. To resolve the identity of the Walker B residue in MglA, we carried out a comprehensive analysis of Mg2+ coordination on P-loop NTPase structures. Atoms in the octahedral coordination of Mg2+ and their interactions comprise a network including water molecules, Walker A, Walker B and switch motifs of P-loop NTPases. Based on the conserved geometry of Mg2+ coordination, we confirm that a conserved aspartate functions as the Walker B residue of MglA, and validate it through mutagenesis and biochemical characterization. Location of the newly identified aspartate is spatially equivalent to the Walker B residue of the ASCE division of P-loop NTPases. Furthermore, similar to the allosteric regulation of the Walker B aspartate conformation in MglA, we identify protein families in which large conformational changes involving Walker B motif potentially function as allosteric regulators. The study unravels conserved features of Mg2+ coordination among divergent families of P-loop NTPases, especially between ancient Ras-like GTPases and ASCE family of ATPases. The conserved geometric features provide a foundation for design of nucleotide-hydrolyzing enzymes.


Assuntos
Domínio AAA/fisiologia , Proteínas AAA/metabolismo , GTP Fosfo-Hidrolases/química , Células Procarióticas/metabolismo , Proteínas ras/química , Proteínas AAA/genética , Evolução Molecular , GTP Fosfo-Hidrolases/genética , Modelos Moleculares , Nucleosídeo-Trifosfatase/metabolismo , Conformação Proteica , Proteínas ras/genética
6.
Curr Biol ; 30(6): R251-R257, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32208144

RESUMO

In this Primer, Seraphim and Houry highlight the structural features and functional diversity of AAA+ proteins and summarise our current knowledge of the molecular mechanisms driving the activities of these proteins.


Assuntos
Proteínas AAA/química , Proteínas AAA/fisiologia , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo
7.
Biomolecules ; 10(3)2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32106553

RESUMO

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA+ ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA+ domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA+ domain of a bEBP bound to RNAP-σ54-promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA+ proteins utilises ATPase activities to allow σ54-dependent transcription initiation.


Assuntos
Proteínas AAA/metabolismo , Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas AAA/química , Proteínas AAA/genética , Trifosfato de Adenosina/metabolismo , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética
8.
Elife ; 92020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-31999255

RESUMO

The AAA protein Msp1 extracts mislocalized tail-anchored membrane proteins and targets them for degradation, thus maintaining proper cell organization. How Msp1 selects its substrates and firmly engages them during the energetically unfavorable extraction process remains a mystery. To address this question, we solved cryo-EM structures of Msp1-substrate complexes at near-atomic resolution. Akin to other AAA proteins, Msp1 forms hexameric spirals that translocate substrates through a central pore. A singular hydrophobic substrate recruitment site is exposed at the spiral's seam, which we propose positions the substrate for entry into the pore. There, a tight web of aromatic amino acids grips the substrate in a sequence-promiscuous, hydrophobic milieu. Elements at the intersubunit interfaces coordinate ATP hydrolysis with the subunits' positions in the spiral. We present a comprehensive model of Msp1's mechanism, which follows general architectural principles established for other AAA proteins yet specializes Msp1 for its unique role in membrane protein extraction.


Assuntos
Proteínas AAA/química , Proteínas Fúngicas/química , Proteínas de Membrana/química , Leveduras/metabolismo , Proteínas AAA/metabolismo , Microscopia Crioeletrônica , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Conformação Proteica , Transporte Proteico
9.
Nat Rev Mol Cell Biol ; 21(1): 43-58, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31754261

RESUMO

ATPases associated with diverse cellular activities (AAA+ proteins) are macromolecular machines that convert the chemical energy contained in ATP molecules into powerful mechanical forces to remodel a vast array of cellular substrates, including protein aggregates, macromolecular complexes and polymers. AAA+ proteins have key functionalities encompassing unfolding and disassembly of such substrates in different subcellular localizations and, hence, power a plethora of fundamental cellular processes, including protein quality control, cytoskeleton remodelling and membrane dynamics. Over the past 35 years, many of the key elements required for AAA+ activity have been identified through genetic, biochemical and structural analyses. However, how ATP powers substrate remodelling and whether a shared mechanism underlies the functional diversity of the AAA+ superfamily were uncertain. Advances in cryo-electron microscopy have enabled high-resolution structure determination of AAA+ proteins trapped in the act of processing substrates, revealing a conserved core mechanism of action. It has also become apparent that this common mechanistic principle is structurally adjusted to carry out a diverse array of biological functions. Here, we review how substrate-bound structures of AAA+ proteins have expanded our understanding of ATP-driven protein remodelling.


Assuntos
Proteínas AAA/química , Proteínas AAA/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Microscopia Crioeletrônica , Humanos , Hidrólise , Modelos Moleculares , Conformação Proteica
10.
Cell Chem Biol ; 26(9): 1263-1273.e5, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31257183

RESUMO

Drug-like inhibitors are often designed by mimicking cofactor or substrate interactions with enzymes. However, as active sites are comprised of conserved residues, it is difficult to identify the critical interactions needed to design selective inhibitors. We are developing an approach, named RADD (resistance analysis during design), which involves engineering point mutations in the target to generate active alleles and testing compounds against them. Mutations that alter compound potency identify residues that make key interactions with the inhibitor and predict target-binding poses. Here, we apply this approach to analyze how diaminotriazole-based inhibitors bind spastin, a microtubule-severing AAA (ATPase associated with diverse cellular activities) protein. The distinct binding poses predicted for two similar inhibitors were confirmed by a series of X-ray structures. Importantly, our approach not only reveals how selective inhibition of the target can be achieved but also identifies resistance-conferring mutations at the early stages of the design process.


Assuntos
Engenharia de Proteínas/métodos , Espastina/efeitos dos fármacos , Espastina/genética , Proteínas AAA/genética , Adenosina Trifosfatases/metabolismo , Amitrol (Herbicida)/química , Fenômenos Bioquímicos , Domínio Catalítico , Cristalografia por Raios X/métodos , Desenho de Fármacos , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Mutação Puntual/genética , Espastina/antagonistas & inibidores , Triazóis/química , Tubulina (Proteína)/química
11.
Curr Opin Chem Biol ; 50: 45-54, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30913482

RESUMO

The AAA proteins are a family of enzymes that play key roles in diverse dynamic cellular processes, ranging from proteostasis to directional intracellular transport. Dysregulation of AAA proteins has been linked to several diseases, including cancer, suggesting a possible therapeutic role for inhibitors of these enzymes. In the past decade, new chemical probes have been developed for AAA proteins including p97, dynein, midasin, and ClpC1. In this review, we discuss how these compounds have been used to study the cellular functions and conformational dynamics of AAA proteins. We discuss future directions for inhibitor development and early efforts to utilize AAA protein inhibitors in the clinical setting.


Assuntos
Proteínas AAA/química , Proteínas AAA/fisiologia , Sondas Moleculares , Preparações Farmacêuticas , Proteínas AAA/metabolismo , Humanos , Organelas/metabolismo , Conformação Proteica
12.
Sci Rep ; 9(1): 712, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679587

RESUMO

Despite recent advances in understanding the biogenesis of iron-sulfur (Fe-S) proteins, most studies focused on aerobic bacteria as model organisms. Accordingly, multiple players have been proposed to participate in the Fe-S delivery step to apo-target proteins, but critical gaps exist in the knowledge of Fe-S proteins biogenesis in anaerobic organisms. Mrp/NBP35 ATP-binding proteins are a subclass of the soluble P-loop containing nucleoside triphosphate hydrolase superfamily (P-loop NTPase) known to bind and transfer Fe-S clusters in vitro. Here, we report investigations of a novel atypical two-domain Mrp/NBP35 ATP-binding protein named MrpORP associating a P-loop NTPase domain with a dinitrogenase iron-molybdenum cofactor biosynthesis domain (Di-Nase). Characterization of full length MrpORP, as well as of its two domains, showed that both domains bind Fe-S clusters. We provide in vitro evidence that the P-loop NTPase domain of the MrpORP can efficiently transfer its Fe-S cluster to apo-target proteins of the ORange Protein (ORP) complex, suggesting that this novel protein is involved in the maturation of these Fe-S proteins. Last, we showed for the first time, by fluorescence microscopy imaging a polar localization of a Mrp/NBP35 protein.


Assuntos
Proteínas de Bactérias/metabolismo , Desulfovibrio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Enxofre/metabolismo , Proteínas AAA/genética , Proteínas AAA/metabolismo , Proteínas de Bactérias/genética , Citosol , Desulfovibrio/classificação , Desulfovibrio/genética , Proteínas de Ligação ao GTP/genética , Proteínas Ferro-Enxofre/genética , Molibdoferredoxina/metabolismo , Nitrogenase/genética , Nitrogenase/metabolismo , Ligação Proteica , Domínios Proteicos
13.
Elife ; 72018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30047865

RESUMO

The innate immune sensor retinoic acid-inducible gene I (RIG-I) detects cytosolic viral RNA and requires a conformational change caused by both ATP and RNA binding to induce an active signaling state and to trigger an immune response. Previously, we showed that ATP hydrolysis removes RIG-I from lower-affinity self-RNAs (Lässig et al., 2015), revealing how ATP turnover helps RIG-I distinguish viral from self-RNA and explaining why a mutation in a motif that slows down ATP hydrolysis causes the autoimmune disease Singleton-Merten syndrome (SMS). Here we show that a different, mechanistically unexplained SMS variant, C268F, which is localized in the ATP-binding P-loop, can signal independently of ATP but is still dependent on RNA. The structure of RIG-I C268F in complex with double-stranded RNA reveals that C268F helps induce a structural conformation in RIG-I that is similar to that induced by ATP. Our results uncover an unexpected mechanism to explain how a mutation in a P-loop ATPase can induce a gain-of-function ATP state in the absence of ATP.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Doenças da Aorta/genética , Proteína DEAD-box 58/química , Hipoplasia do Esmalte Dentário/genética , Metacarpo/anormalidades , Doenças Musculares/genética , Odontodisplasia/genética , Osteoporose/genética , Calcificação Vascular/genética , Proteínas AAA/química , Proteínas AAA/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Doenças da Aorta/enzimologia , Doenças da Aorta/patologia , Citosol/virologia , Proteína DEAD-box 58/genética , Hipoplasia do Esmalte Dentário/enzimologia , Hipoplasia do Esmalte Dentário/patologia , Humanos , Hidrólise , Imunidade Inata/genética , Metacarpo/enzimologia , Metacarpo/patologia , Doenças Musculares/enzimologia , Doenças Musculares/patologia , Mutação , Odontodisplasia/enzimologia , Odontodisplasia/patologia , Osteoporose/enzimologia , Osteoporose/patologia , Ligação Proteica , Conformação Proteica , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética , Calcificação Vascular/enzimologia , Calcificação Vascular/patologia
14.
Cell Res ; 28(3): 296-306, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29451229

RESUMO

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.


Assuntos
Proteínas AAA/metabolismo , Cálcio/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Doenças Neurodegenerativas/enzimologia , Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Canais de Cálcio/metabolismo , Humanos , Metaloendopeptidases/genética , Camundongos , Mitocôndrias/genética , Modelos Animais , Células de Purkinje/enzimologia , Paraplegia Espástica Hereditária/genética , Ataxias Espinocerebelares/genética
15.
Mol Genet Genomics ; 293(1): 17-31, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28900732

RESUMO

STAND P-loop NTPase is the common weapon used by plant and other organisms from all three kingdoms of life to defend themselves against pathogen invasion. The purpose of this study is to review comprehensively the latest finding of plant STAND P-loop NTPase related to their genomic distribution, evolution, and their mechanism of action. Earlier, the plant STAND P-loop NTPase known to be comprised of only NBS-LRRs/AP-ATPase/NB-ARC ATPase. However, recent finding suggests that genome of early green plants comprised of two types of STAND P-loop NTPases: (1) mammalian NACHT NTPases and (2) NBS-LRRs. Moreover, YchF (unconventional G protein and members of P-loop NTPase) subfamily has been reported to be exceptionally involved in biotic stress (in case of Oryza sativa), thereby a novel member of STAND P-loop NTPase in green plants. The lineage-specific expansion and genome duplication events are responsible for abundance of plant STAND P-loop NTPases; where "moderate tandem and low segmental duplication" trajectory followed in majority of plant species with few exception (equal contribution of tandem and segmental duplication). Since the past decades, systematic research is being investigated into NBS-LRR function supported the direct recognition of pathogen or pathogen effectors by the latest models proposed via 'integrated decoy' or 'sensor domains' model. Here, we integrate the recently published findings together with the previous literature on the genomic distribution, evolution, and distinct models proposed for functional molecular mechanism of plant STAND P-loop NTPases.


Assuntos
Proteínas AAA/genética , Resistência à Doença/genética , Evolução Molecular , Genoma de Planta/genética , Adenosina Trifosfatases/genética , Genômica , Oryza/enzimologia , Oryza/genética , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Homologia de Sequência de Aminoácidos
16.
Fungal Genet Biol ; 95: 1-12, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27473887

RESUMO

The peroxisome plays an essential role in eukaryotic cellular metabolism, including ß-oxidation of fatty acids and detoxification of hydrogen peroxide. However, its functions in the important fungal pathogen, C. albicans, remain to be investigated. In this study, we identified a homologue of Saccharomyces cerevisiae peroxisomal protein Pex1 in this pathogen, and explored its functions in stress tolerance. Fluorescence observation revealed that C. albicans Pex1 was localized in the peroxisomes, and its loss led to the defect in peroxisome formation. Interestingly, the pex1Δ/Δ mutant had increased tolerance to oxidative stress, which was neither associated with the Cap1 pathway, nor related to the altered distribution of catalase. However, under oxidative stress, the pex1Δ/Δ mutant showed increased expression of autophagy-related genes, with enhanced cytoplasm-to-vacuole transport and degradation of the autophagy markers Atg8 and Lap41. Moreover, the double mutants pex1Δ/Δatg8Δ/Δ and pex1Δ/Δatg1Δ/Δ, both of which were defective in autophagy and peroxisome formation, showed remarkable attenuated tolerance to oxidative stress. These results indicated that autophagy is involved in resistance to oxidative stress in pex1Δ/Δ mutant. Taken together, this study provides evidence that the peroxisomal protein Pex1 regulates oxidative stress tolerance in an autophagy-dependent manner in C. albicans.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Candida albicans/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Estresse Oxidativo/fisiologia , Peroxissomos/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas AAA , ATPases Associadas a Diversas Atividades Celulares/genética , Autofagia/genética , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Catalase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Peróxido de Hidrogênio/metabolismo , Macrófagos/microbiologia , Proteínas de Membrana/genética , Mutação , Peroxissomos/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico , Vacúolos/metabolismo , Virulência
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