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1.
Nat Commun ; 11(1): 2489, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427831

RESUMO

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

2.
Nat Commun ; 11(1): 769, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034139

RESUMO

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH.


Assuntos
Histidina Quinase/química , Histidina Quinase/metabolismo , Thermotoga maritima/enzimologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Histidina Quinase/genética , Concentração de Íons de Hidrogênio , Modelos Biológicos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação , Conformação Proteica , Thermotoga maritima/genética , Transativadores/química , Transativadores/metabolismo
3.
Methods Mol Biol ; 2077: 121-140, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31707656

RESUMO

Autophosphorylation of histidine kinases (HK) is the first step for signal transduction in bacterial two-component signalling systems. As HKs dimerize, the His residue is phosphorylated in cis or trans depending on whether the ATP molecule used in the reaction is bound to the same or the neighboring subunit, respectively. The cis or trans autophosphorylation results from an alternative directionality in the connection between helices α1 and α2 in the HK DHp domain, in such a way that α2 could be oriented almost 90° counterclockwise or clockwise with respect to α1. Sequence and length variability of this connection appears to lie behind the different directionality and is implicated in partner recognition with the response regulator (RR), highlighting its importance in signal transduction. Despite this mechanistic difference, HK autophosphorylation appears to be universal, involving conserved residues neighboring the phosphoacceptor His residue. Herein, we describe a simple protocol to determine both autophosphorylation directionality of HKs and the roles of the catalytic residues in these protein kinases.

4.
IUCrJ ; 5(Pt 6): 765-779, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30443360

RESUMO

Basement membranes are extracellular structures of epithelia and endothelia that have collagen IV scaffolds of triple α-chain helical protomers that associate end-to-end, forming networks. The molecular mechanisms by which the noncollagenous C-terminal domains of α-chains direct the selection and assembly of the α1α2α1 and α3α4α5 hetero-oligomers found in vivo remain obscure. Autoantibodies against the noncollagenous domains of the α3α4α5 hexamer or mutations therein cause Goodpasture's or Alport's syndromes, respectively. To gain further insight into oligomer-assembly mechanisms as well as into Goodpasture's and Alport's syndromes, crystal structures of non-collagenous domains produced by recombinant methods were determined. The spontaneous formation of canonical homohexamers (dimers of trimers) of these domains of the α1, α3 and α5 chains was shown and the components of the Goodpasture's disease epitopes were viewed. Crystal structures of the α2 and α4 non-collagenous domains generated by recombinant methods were also determined. These domains spontaneously form homo-oligomers that deviate from the canonical architectures since they have a higher number of subunits (dimers of tetramers and of hexamers, respectively). Six flexible structural motifs largely explain the architectural variations. These findings provide insight into noncollagenous domain folding, while supporting the in vivo operation of extrinsic mechanisms for restricting the self-assembly of noncollagenous domains. Intriguingly, Alport's syndrome missense mutations concentrate within the core that nucleates the folding of the noncollagenous domain, suggesting that this syndrome, when owing to missense changes, is a folding disorder that is potentially amenable to pharmacochaperone therapy.

5.
J Phys Chem Lett ; 9(16): 4522-4526, 2018 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-30044106

RESUMO

The pursuit of novel functional building blocks for the emerging field of quantum computing is one of the most appealing topics in the context of quantum technologies. Herein we showcase the urgency of introducing peptides as versatile platforms for quantum computing. In particular, we focus on lanthanide-binding tags, originally developed for the study of protein structure. We use pulsed electronic paramagnetic resonance to demonstrate quantum coherent oscillations in both neodymium and gadolinium peptidic qubits. Calculations based on density functional theory followed by a ligand field analysis indicate the possibility of influencing the nature of the spin qubit states by means of controlled changes in the peptidic sequence. We conclude with an overview of the challenges and opportunities opened by this interdisciplinary field.


Assuntos
Metaloproteínas/química , Peptídeos/química , Teoria Quântica , Cátions/química , Espectroscopia de Ressonância de Spin Eletrônica , Elementos da Série dos Lantanídeos/química , Modelos Químicos
6.
Nucleic Acids Res ; 46(1): 456-472, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29186528

RESUMO

The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3-. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a ß5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic.


Assuntos
Proteínas de Bactérias/química , DNA/química , Conformação de Ácido Nucleico , Domínios Proteicos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Homologia de Sequência de Aminoácidos
7.
Nat Commun ; 5: 3258, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24500224

RESUMO

Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Domínio Catalítico , Análise Mutacional de DNA , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Histidina Quinase , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Fosforilação , Conformação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Relação Estrutura-Atividade
8.
Structure ; 21(9): 1636-47, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23954504

RESUMO

Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, Escherichia coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes and a systematic mutagenesis study reveal how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions and protein evolution and for the design of novel protein interfaces.


Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/enzimologia , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais
9.
Curr Opin Struct Biol ; 20(6): 763-71, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20951027

RESUMO

Two-component systems, composed of a homodimeric histidine kinase (HK) and a response regulator (RR), are major signal transduction devices in bacteria. Typically the signal triggers HK autophosphorylation at one His residue, followed by phosphoryl transfer from the phospho-His to an Asp residue in the RR. Signal extinction frequently involves phospho-RR dephosphorylation by a phosphatase activity of the HK. Our understanding of these reactions and of the determinants of partner specificity among HK-RR couples has been greatly increased by recent crystal structures and biochemical experiments on HK-RR complexes. Cis-autophosphorylation (one subunit phosphorylates itself) occurs in some HKs while trans-autophosphorylation takes place in others. We review and integrate this new information, discuss the mechanism of the three reactions and propose a model for transmembrane signaling by these systems.


Assuntos
Transdução de Sinais , Citoplasma/metabolismo , Histidina Quinase , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/metabolismo
10.
Cell ; 139(2): 325-36, 2009 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-19800110

RESUMO

The chief mechanism used by bacteria for sensing their environment is based on two conserved proteins: a sensor histidine kinase (HK) and an effector response regulator (RR). The signal transduction process involves highly conserved domains of both proteins that mediate autokinase, phosphotransfer, and phosphatase activities whose output is a finely tuned RR phosphorylation level. Here, we report the structure of the complex between the entire cytoplasmic portion of Thermotoga maritima class I HK853 and its cognate, RR468, as well as the structure of the isolated RR468, both free and BeF(3)(-) bound. Our results provide insight into partner specificity in two-component systems, recognition of the phosphorylation state of each partner, and the catalytic mechanism of the phosphatase reaction. Biochemical analysis shows that the HK853-catalyzed autokinase reaction proceeds by a cis autophosphorylation mechanism within the HK subunit. The results suggest a model for the signal transduction mechanism in two-component systems.


Assuntos
Proteínas de Bactérias/química , Transdução de Sinais , Thermotoga maritima/química , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Thermotoga maritima/metabolismo
11.
Biochemistry ; 46(26): 7713-27, 2007 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-17559195

RESUMO

Allosteric interactions regulate substrate channeling in Salmonella typhimurium tryptophan synthase. The channeling of indole between the alpha- and beta-sites via the interconnecting 25 A tunnel is regulated by allosteric signaling arising from binding of ligand to the alpha-site, and covalent reaction of l-Ser at the beta-site. This signaling switches the alpha- and beta-subunits between open conformations of low activity and closed conformations of high activity. Our objective is to synthesize and characterize new classes of alpha-site ligands (ASLs) that mimic the binding of substrates, 3-indole-d-glycerol 3'-phosphate (IGP) or d-glyceraldehyde 3-phosphate (G3P), for use in the investigation of alpha-site-beta-site interactions. The new synthesized IGP analogues contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups. The G3P analogue, thiophosphoglycolohydroxamate, contains a hydroxamic acid group linked to a thiophosphate moiety. Crystal structures of the internal aldimine complexed with G3P and with three of the new ASLs are presented. These structural and solution studies of the ASL complexes with the internal aldimine form of the enzyme establish the following. (1) ASL binding occurs with high specificity and relatively high affinities at the alpha-site. (2) Binding of the new ASLs slows the entry of indole analogues into the beta-site by blocking the tunnel opening at the alpha-site. (3) ASL binding stabilizes the closed conformations of the beta-subunit for the alpha-aminoacrylate and quinonoid forms of the enzyme. (4) The new ASLs exhibit allosteric properties that parallel the behaviors of IGP and G3P.


Assuntos
Regulação Alostérica/fisiologia , Triptofano Sintase/química , Naftalenossulfonato de Anilina/química , Sítios de Ligação , Gliceraldeído 3-Fosfato/metabolismo , Glicerofosfatos/metabolismo , Cinética , Modelos Químicos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Salmonella typhimurium/enzimologia , Serina/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Triptofano Sintase/metabolismo
12.
Biochemistry ; 46(26): 7728-39, 2007 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-17559231

RESUMO

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.


Assuntos
Regulação Alostérica/fisiologia , Indóis/metabolismo , Triptofano Sintase/metabolismo , Sítios de Ligação , Dicroísmo Circular , Cristalização , Cinética , Conformação Proteica , Espectrofotometria Ultravioleta , Triptofano Sintase/química
13.
Biochim Biophys Acta ; 1774(5): 603-9, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17478132

RESUMO

Two-component signal transduction systems, comprised of histidine kinase and its cognate response regulator, are the predominant mechanism by which microorganisms sense and respond to changes in many different environmental conditions. Different Thermotoga maritima histidine kinases have been used as prototypes; among them, the orphan TM0853 has been presented as a structural model of class I histidine kinases. We used phosphotransfer assays to identify TM0468 as the partner response regulator of TM0853. Since full-length TM0853 can be produced as a soluble protein in Escherichia coli, it was used to analyze the union stoichiometry in an intact two-component system for the first time. We demonstrate that TM0853, or its cytoplasmic catalytic portion, form a 1:1 complex with TM0468 with native PAGE. The complex band is unique, even in the presence of an excess of each individual protein, indicating that the union is cooperative. We corroborated these findings by using ultracentrifugation assays. Therefore, we propose that the general mode of interaction in an orthodox two-component system may be the stoichiometric and cooperative complex between a dimeric histidine kinase and two response regulators. Finally, we have been able to produce protein crystals of the complex between the cytoplasmic portion of TM0853 and TM0468 that diffract to 2.8 A Bragg spacing.


Assuntos
Thermotoga maritima/química , Sequência de Bases , Cristalização , Primers do DNA , Histidina Quinase , Conformação Proteica , Proteínas Quinases/metabolismo , Thermotoga maritima/enzimologia , Ultracentrifugação
14.
Anal Bioanal Chem ; 374(2): 262-8, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12324847

RESUMO

A highly sensitive and specific indirect enzyme-linked immunosorbent assay is described for Silvex, 2-(2,4,5 trichlorophenoxy)propionic acid, (2,4,5-TP). One specific feature of the immunoassay is the use of simple chemical activation of chlorophenoxy acids to prepare both the immunizing and coating conjugates. The assay is based on the use of polyclonal antibodies raised against 2,4,5-TP, and a peroxidase-labeled secondary antibody for colorimetric detection. The effect of different chemical conditions (pH, and salt and detergent concentration) on immunoassay performance has been studied. Under the best conditions the least detectable dose and the sensitivity (IC(50)) for 2,4,5-TP were 0.05 micro g L(-1) and 0.80 micro g L(-1), respectively. The optimized immunoassay was also highly specific, showing little (6.9% for 2,4,5-T) or no cross-reactivity with other similar herbicides. The assay was used to determine 2,4,5-TP in water and soils. The excellent recoveries obtained (mean values ranging between 89% and 104%) make this immunoassay a suitable screening method for either environmental monitoring or laboratory quantification of 2,4,5-TP.


Assuntos
Ácido 2,4,5-Triclorofenoxiacético/análogos & derivados , Ácido 2,4,5-Triclorofenoxiacético/análise , Ensaio de Imunoadsorção Enzimática/métodos , Poluentes Químicos da Água/análise , Animais , Feminino , Coelhos
15.
Biochemistry ; 41(31): 9991-10001, 2002 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-12146963

RESUMO

Reactions catalyzed by the beta-subunits of the tryptophan synthase alpha(2)beta(2) complex involve multiple covalent transformations facilitated by proton transfers between the coenzyme, the reacting substrates, and acid-base catalytic groups of the enzyme. However, the UV/Vis absorbance spectra of covalent intermediates formed between the pyridoxal 5'-phosphate coenzyme (PLP) and the reacting substrate are remarkably pH-independent. Furthermore, the alpha-aminoacrylate Schiff base intermediate, E(A-A), formed between L-Ser and enzyme-bound PLP has an unusual spectrum with lambda(max) = 350 nm and a shoulder extending to greater than 500 nm. Other PLP enzymes that form E(A-A) species exhibit intense bands with lambda(max) approximately 460-470 nm. To further investigate this unusual tryptophan synthase E(A-A) species, these studies examine the kinetics of H(+) release in the reaction of L-Ser with the enzyme using rapid kinetics and the H(+) indicator phenol red in solutions weakly buffered by substrate L-serine. This work establishes that the reaction of L-Ser with tryptophan synthase gives an H(+) release when the external aldimine of L-Ser, E(Aex(1)), is converted to E(A-A). This same H(+) release occurs in the reaction of L-Ser plus the indole analogue, aniline, in a step that is rate-determining for the appearance of E(Q)(Aniline). We propose that the kinetic and spectroscopic properties of the L-Ser reaction with tryptophan synthase reflect a mechanism wherein the kinetically detected proton release arises from conversion of an E(Aex(1)) species protonated at the Schiff base nitrogen to an E(A-A) species with a neutral Schiff base nitrogen. The mechanistic and conformational implications of this transformation are discussed.


Assuntos
Prótons , Triptofano Sintase/metabolismo , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Tampões (Química) , Catálise , Césio/química , Fenolsulfonaftaleína/química , Serina/química , Serina/metabolismo , Sódio/química , Soluções , Espectrofotometria Ultravioleta , Triptofano Sintase/química
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