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1.
J Am Chem Soc ; 145(38): 20985-21001, 2023 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-37707433

RESUMO

Adaptation of avian influenza RNA polymerase (FluPol) to human cells requires mutations on the 627-NLS domains of the PB2 subunit. The E627K adaptive mutation compensates a 33-amino-acid deletion in the acidic intrinsically disordered domain of the host transcription regulator ANP32A, a deletion that restricts FluPol activity in mammalian cells. The function of ANP32A in the replication transcription complex and in particular its role in host restriction remains poorly understood. Here we characterize ternary complexes formed between ANP32A, FluPol, and the viral nucleoprotein, NP, supporting the putative role of ANP32A in shuttling NP to the replicase complex. We demonstrate that while FluPol and NP can simultaneously bind distinct linear motifs on avian ANP32A, the deletion in the shorter human ANP32A blocks this mode of colocalization. NMR reveals that NP and human-adapted FluPol, containing the E627 K mutation, simultaneously bind the identical extended linear motif on human ANP32A in an electrostatically driven, highly dynamic and multivalent ternary complex. This study reveals a probable molecular mechanism underlying host adaptation, whereby E627K, which enhances the basic surface of the 627 domain, is selected to confer the necessary multivalent properties to allow ANP32A to colocalize NP and FluPol in human cells.


Assuntos
Influenza Aviária , Animais , Humanos , Nucleotidiltransferases , Aminoácidos , Mutação , Probabilidade , Mamíferos , Proteínas Nucleares , Proteínas de Ligação a RNA/genética
2.
Chem Rev ; 122(10): 9331-9356, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35446534

RESUMO

Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.


Assuntos
Proteínas Intrinsicamente Desordenadas , Humanos , Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Termodinâmica
3.
Sci Adv ; 8(3): eabm4034, 2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35044811

RESUMO

The processes of genome replication and transcription of SARS-CoV-2 represent important targets for viral inhibition. Betacoronaviral nucleoprotein (N) is a highly dynamic cofactor of the replication-transcription complex (RTC), whose function depends on an essential interaction with the amino-terminal ubiquitin-like domain of nsp3 (Ubl1). Here, we describe this complex (dissociation constant - 30 to 200 nM) at atomic resolution. The interaction implicates two linear motifs in the intrinsically disordered linker domain (N3), a hydrophobic helix (219LALLLLDRLNQL230) and a disordered polar strand (243GQTVTKKSAAEAS255), that mutually engage to form a bipartite interaction, folding N3 around Ubl1. This results in substantial collapse in the dimensions of dimeric N, forming a highly compact molecular chaperone, that regulates binding to RNA, suggesting a key role of nsp3 in the association of N to the RTC. The identification of distinct linear motifs that mediate an important interaction between essential viral factors provides future targets for development of innovative strategies against COVID-19.

4.
Angew Chem Int Ed Engl ; 61(1): e202109961, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34750927

RESUMO

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade-unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS-8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature-dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/síntese química , Animais , Géis/química , Proteínas Intrinsicamente Desordenadas/química , Estresse Fisiológico , Tardígrados
5.
Front Mol Biosci ; 8: 653148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34041264

RESUMO

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

6.
Nat Commun ; 11(1): 3656, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694517

RESUMO

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.


Assuntos
Proteínas Aviárias/ultraestrutura , Virus da Influenza A Subtipo H5N1/patogenicidade , Proteínas Nucleares/ultraestrutura , Domínios Proteicos/genética , Proteínas de Ligação a RNA/ultraestrutura , RNA Polimerase Dependente de RNA/ultraestrutura , Proteínas Virais/ultraestrutura , Animais , Proteínas Aviárias/metabolismo , Aves/virologia , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/metabolismo , Influenza Aviária/virologia , Influenza Humana/virologia , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Ligação Proteica/genética , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Especificidade da Espécie , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
7.
Chemistry ; 23(53): 13010-13014, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28763125

RESUMO

Accumulation of α-synuclein (αSyn) aggregates constitutes the hallmark of synucleinopathies including Parkinson's disease. However, many steps from the innocuous, monomeric αSyn toward misfolded oligomers and fibrillar species remain unclear. Here, we show that αSyn can form in solution α-helical oligomers, which are off-pathway to fibrillization, through interaction with the tetrapyrrole phthalocyanine tetrasulfonate. Chemical cross-linking combined with mass spectrometry reveals a large number of intermolecular cross-links along the entire αSyn sequence in the phthalocyanine tetrasulfonate-stabilized αSyn oligomers. Our study suggests that stabilization of structured oligomers by small molecules provides a viable strategy to interfere with αSyn fibrillization.


Assuntos
alfa-Sinucleína/química , Sequência de Aminoácidos , Complexos de Coordenação/química , Reagentes de Ligações Cruzadas , Espectrometria de Massas/métodos , Doença de Parkinson/metabolismo , Ligação Proteica , Dobramento de Proteína , Multimerização Proteica , Rutênio/química , Solubilidade
8.
Angew Chem Int Ed Engl ; 54(1): 336-9, 2015 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-25293958

RESUMO

Paramagnetic effects provide unique information about the structure and dynamics of biomolecules. We developed a method in which the lanthanoid tag is not directly attached to the protein of interest, but instead to a "reporter" protein, which binds and then transmits paramagnetic information to the target. The designed method allows access to a large number of paramagnetic restraints and residual dipolar couplings produced from independent molecular alignments in high-molecular-weight proteins with unknown 3D structure.


Assuntos
Elementos da Série dos Lantanídeos/química , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Escherichia coli/química , Humanos , Proteínas Ligantes de Maltose/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Terciária de Proteína , Ubiquitina/química
9.
J Virol ; 87(21): 11894-907, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23986592

RESUMO

Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.


Assuntos
Motivos de Aminoácidos , Baculoviridae/fisiologia , Colesterol/metabolismo , Proteínas Virais de Fusão/metabolismo , Ligação Viral , Linhagem Celular , Endocitose , Humanos , Proteínas Virais de Fusão/genética
10.
Mol Biol Evol ; 30(9): 2024-34, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23800623

RESUMO

Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (ßα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (ßα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism.


Assuntos
Aldose-Cetose Isomerases/genética , Proteínas de Bactérias/genética , Corynebacterium/genética , Evolução Molecular , Transferência Genética Horizontal , Mycobacterium/genética , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Corynebacterium/classificação , Corynebacterium/enzimologia , Cristalografia por Raios X , Histidina/biossíntese , Histidina/genética , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium/classificação , Mycobacterium/enzimologia , Óperon , Filogenia , Estrutura Secundária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Triptofano/biossíntese , Triptofano/genética
11.
Protein Sci ; 19(3): 535-43, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20066665

RESUMO

A good model to experimentally explore evolutionary hypothesis related to enzyme function is the ancient-like dual-substrate (beta alpha)(8) phosphoribosyl isomerase A (PriA), which takes part in both histidine and tryptophan biosynthesis in Streptomyces coelicolor and related organisms. In this study, we determined the Michaelis-Menten enzyme kinetics for both isomerase activities in wild-type PriA from S. coelicolor and in selected single-residue monofunctional mutants, identified after Escherichia coli in vivo complementation experiments. Structural and functional analyses of a hitherto unnoticed residue contained on the functionally important beta --> alpha loop 5, namely, Arg(139), which was postulated on structural grounds to be important for the dual-substrate specificity of PriA, is presented for the first time. Indeed, enzyme kinetics analyses done on the mutant variants PriA_Ser(81)Thr and PriA_Arg(139)Asn showed that these residues, which are contained on beta --> alpha loops and in close proximity to the N-terminal phosphate-binding site, are essential solely for the phosphoribosyl anthranilate isomerase activity of PriA. Moreover, analysis of the X-ray crystallographic structure of PriA_Arg(139)Asn elucidated at 1.95 A herein strongly implicates the occurrence of conformational changes in this beta --> alpha loop as a major structural feature related to the evolution of the dual-substrate specificity of PriA. It is suggested that PriA has evolved by tuning a fine energetic balance that allows the sufficient degree of structural flexibility needed for accommodating two topologically dissimilar substrates--within a bifunctional and thus highly constrained active site--without compromising its structural stability.


Assuntos
Aldose-Cetose Isomerases/química , Evolução Molecular , Streptomyces coelicolor/enzimologia , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Arginina/química , Asparagina/química , Domínio Catalítico , Cristalografia por Raios X , Cinética , Conformação Proteica , Estrutura Secundária de Proteína , Análise de Sequência de Proteína , Serina/química , Treonina/química
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