RESUMO
Vibrio cholerae is the causative agent of the diarrheal disease cholera, for which biofilm communities are considered to be environmental reservoirs. In endemic regions, and after algal blooms, which may result from phosphate enrichment following agricultural runoff, the bacterium is released from biofilms resulting in seasonal disease outbreaks. However, the molecular mechanism by which V. cholerae senses its environment and switches lifestyles from the biofilm-bound state to the planktonic state is largely unknown. Here, we report that the major biofilm scaffolding protein RbmA undergoes autocatalytic proteolysis via a phosphate-dependent induced proximity activation mechanism. Furthermore, we show that RbmA mutants that are defective in autoproteolysis cause V. cholerae biofilms to grow larger and mechanically stronger, correlating well with the observation that RbmA stability directly affects microbial community homeostasis and rheological properties. In conclusion, our biophysical study characterizes a novel phosphate-dependent breakdown pathway of RbmA, while microbiological data suggest a new, sensory role of this biofilm scaffolding element.
Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Compostos de Magnésio/farmacologia , Fosfatos/farmacologia , Proteólise , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
Photolyases and cryptochromes form an almost ubiquitous family of blue light photoreceptors involved in the repair and maintenance of DNA integrity or regulatory control. We found that one cryptochrome from the green alga Chlamydomonas reinhardtii (CraCRY) is capable of both, control of transcript levels and the sexual cycle of the alga in a positive (germination) and negative manner (mating ability), as well as catalyzing the repair of UV-DNA lesions. Its 1.6 Å crystal structure shows besides the FAD chromophore an aromatic tetrad that is indispensable in animal-like type I cryptochromes for light-driven change of their signaling-active redox state and formation of a stable radical pair. Given CraCRY's catalytic activity as (6-4) photolyase in vivo and in vitro, we present the first co-crystal structure of a cryptochrome with duplex DNA comprising a (6-4) pyrimidine-pyrimidone lesion. This 2.9 Å structure reveals a distinct conformation for the catalytic histidine His1, H357, that challenges previous models of a single-photon driven (6-4) photolyase mechanism.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Criptocromos/metabolismo , Reparo do DNA/fisiologia , Desoxirribodipirimidina Fotoliase/metabolismo , Conformação Molecular , Sequência de Aminoácidos , Chlamydomonas reinhardtii/genética , Cristalografia por Raios X , Modelos Moleculares , Oxirredução , Alinhamento de Sequência , Transdução de SinaisRESUMO
Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving "prebinding" of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol µ is a unique member of the X-family with multiple functions and with unusual Mn2+ preference. Here we report that Pol µ not only prebinds MdNTP in a catalytically active conformation but also exerts a Mn2+ over Mg2+ preference at this early stage of catalysis, for various functions: incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the nonhomologous end joining (NHEJ) repair, incorporation of dNTP to an ssDNA, and incorporation of an 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn2+ over Mg2+ preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol µ. Overall, this work provides the structural and mechanistic basis for the long-standing puzzle in the Mn2+ preference of Pol µ and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Manganês/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/isolamento & purificação , Humanos , Manganês/química , Modelos MolecularesRESUMO
Human tumor necrosis factor receptor associated factor (TRAF)-interacting protein, with a forkhead-associated domain (TIFA), is a key regulator of NF-κB activation. It also plays a key role in the activation of innate immunity in response to bacterial infection, through heptose 1,7-bisphosphate (HBP); a metabolite of lipopolysaccharide (LPS). However, the mechanism of TIFA function is largely unexplored, except for the suggestion of interaction with TRAF6. Herein, we provide evidence for direct binding, albeit weak, between TIFA and the TRAF domain of TRAF6, and it is shown that the binding is enhanced for a rationally designed double mutant, TIFA S174Q/M179D. Enhanced binding was also demonstrated for endogenous full-length TRAF6. Furthermore, the structures of the TRAF domain complexes with the consensus TRAF-binding peptides from the Câ terminus of wild-type and S174Q/M179D mutant TIFA, showing salt-bridge formation between residues 177-181 of TIFA and the binding pocket residues of the TRAF domain, were solved. Taken together, the results provide direct evidence and a structural basis for the TIFA-TRAF6 interaction, and show how this important biological function can be modulated.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Fator 6 Associado a Receptor de TNF/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Conformação Proteica , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes' light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical 'aureo box', TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity.
Assuntos
DNA/química , Diatomáceas/genética , Transdução de Sinal Luminoso , Fotorreceptores de Plantas/química , Regulação Alostérica , Sítios de Ligação , Ritmo Circadiano/genética , DNA/genética , DNA/metabolismo , Diatomáceas/metabolismo , Diatomáceas/efeitos da radiação , Expressão Gênica , Cinética , Luz , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Fotorreceptores de Plantas/genética , Fotorreceptores de Plantas/metabolismo , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , TermodinâmicaRESUMO
The estuarine gram-negative rod and human diarrheal pathogen Vibrio cholerae synthesizes a VPS exopolysaccharide-dependent biofilm matrix that allows it to form a 3D structure on surfaces. Proteins associated with the matrix include, RbmA, RbmC, and Bap1. RbmA, a protein whose crystallographic structure suggests two binding surfaces, associates with cells by means of a VPS-dependent mechanism and promotes biofilm cohesiveness and recruitment of cells to the biofilm. Here, we show that RbmA undergoes limited proteolysis within the biofilm. This proteolysis, which is carried out by the hemagglutinin/protease and accessory proteases, yields the 22-kDa C-terminal polypeptide RbmA*. RbmA* remains biofilm-associated. Unlike full-length RbmA, the association of RbmA* with cells is no longer VPS-dependent, likely due to an electropositive surface revealed by proteolysis. We provide evidence that this proteolysis event plays a role in recruitment of VPS(-) cells to the biofilm surface. Based on our findings, we propose that association of RbmA with the matrix reinforces the biofilm structure and leads to limited proteolysis of RbmA to RbmA*. RbmA*, in turn, promotes recruitment of cells that have not yet initiated VPS synthesis to the biofilm surface. The assignment of two functions to RbmA, separated by a proteolytic event that depends on matrix association, dictates an iterative cycle in which reinforcement of recently added biofilm layers precedes the recruitment of new VPS(-) cells to the biofilm.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Vibrio cholerae/genética , Sequência de Aminoácidos , Aderência Bacteriana , Quelantes/química , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Mutação , Polissacarídeos Bacterianos/química , Conformação Proteica , Proteólise , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Transcrição GênicaRESUMO
For host colonization, the human fungal pathogen Candida glabrata is known to utilize a large family of highly related surface-exposed cell wall proteins, the lectin-like epithelial adhesins (Epas). To reveal the structure-function relationships within the entire Epa family, we have performed a large scale functional analysis of the adhesion (A) domains of 17 Epa paralogs in combination with three-dimensional structural studies of selected members with cognate ligands. Our study shows that most EpaA domains exert lectin-like functions and together recognize a wide variety of glycans with terminal galactosides for conferring epithelial cell adhesion. We further identify several conserved and variable structural features within the diverse Epa ligand binding pockets, which affect affinity and specificity. These features rationalize why mere phylogenetic relationships within the Epa family are weak indicators for functional classification and explain how Epa-like adhesins have evolved in C. glabrata and related fungal species.
Assuntos
Candida glabrata/química , Moléculas de Adesão Celular/química , Proteínas Fúngicas/química , Lectinas/química , Polissacarídeos/química , Sequência de Aminoácidos , Sítios de Ligação , Evolução Biológica , Candida glabrata/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Variação Genética , Lectinas/genética , Lectinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Polissacarídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.
Assuntos
Ferro/química , Metaloproteínas/química , Óxido Nítrico/química , Dicroísmo Circular , Cristalografia por Raios X , Metaloproteínas/metabolismo , Modelos Moleculares , Óxido Nítrico/metabolismoRESUMO
The teicoplanin-associated locus regulator (TcaR) regulates gene expression of proteins on the intercellular adhesion (ica) locus involved in staphylococci poly-N-acetylglucosamine biosynthesis. The absence of TcaR increases poly-N-acetylglucosamine production and promotes biofilm formation. Until recently, the mechanism of multiple antibiotic resistance regulator family protein members, such as TcaR, was restricted to binding double-stranded DNA. However, we recently found that TcaR strongly interacts with single-stranded DNA, which is a new role for this family of proteins. In this study, we report Staphylococcus epidermidis TcaR-single-stranded DNA complex structures. Our model suggests that TcaR and single-stranded DNA form a 61-symmetry polymer composed of TcaR dimers with single-stranded DNA that wraps outside the polymer and 12 nt per TcaR dimer. Single-stranded DNA binding to TcaR involves a large conformational change at the DNA binding lobe. Several point mutations involving the single-stranded DNA binding surface validate interactions between single-stranded DNA and TcaR. Our results extend the novel role of multiple antibiotic resistance regulator family proteins in staphylococci.
Assuntos
Proteínas de Bactérias/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Staphylococcus epidermidisRESUMO
The human pathogenic yeast Candida glabrata harbors more than 20 surface-exposed, epithelial adhesins (Epas) for host cell adhesion. The Epa family recognizes host glycans and discriminates between target tissues by their adhesin (A) domains, but a detailed structural basis for ligand-binding specificity of Epa proteins has been lacking so far. In this study, we provide high-resolution crystal structures of the Epa1A domain in complex with different carbohydrate ligands that reveal how host cell mucin-type O-glycans are recognized and allow a structure-guided classification of the Epa family into specific subtypes. Further detailed structural and functional characterization of subtype-switched Epa1 variants shows that specificity is governed by two inner loops, CBL1 and CBL2, involved in calcium binding as well as by three outer loops, L1, L2, and L3. In summary, our study provides the structural basis for promiscuity and specificity of Epa adhesins, which might further contribute to developing anti-adhesive antimycotics and combating Candida colonization.
Assuntos
Candida glabrata/química , Proteínas Fúngicas/química , Lectinas/química , Modelos Moleculares , Família Multigênica/genética , Filogenia , Conformação Proteica , Cálcio/metabolismo , Candida glabrata/fisiologia , Análise por Conglomerados , Biologia Computacional , Cristalografia por Raios X , Fluorescência , Proteínas Fúngicas/genética , Lectinas/genética , Polissacarídeos , Ligação ProteicaRESUMO
Crown ethers are small, cyclic polyethers that have found wide-spread use in phase-transfer catalysis and, to a certain degree, in protein chemistry. Crown ethers readily bind metallic and organic cations, including positively charged amino acid side chains. We elucidated the crystal structures of several protein-crown ether co-crystals grown in the presence of 18-crown-6. We then employed biophysical methods and molecular dynamics simulations to compare these complexes with the corresponding apoproteins and with similar complexes with ring-shaped low-molecular-weight polyethylene glycols. Our studies show that crown ethers can modify protein surface behavior dramatically by stabilizing either intra- or intermolecular interactions. Consequently, we propose that crown ethers can be used to modulate a wide variety of protein surface behaviors, such as oligomerization, domain-domain interactions, stabilization in organic solvents, and crystallization.
Assuntos
Éteres Cíclicos/química , Modelos Anatômicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Propriedades de SuperfícieRESUMO
Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.
Assuntos
Proteínas Arqueais , Reparo do DNA , Desoxirribodipirimidina Fotoliase , Methanosarcina , Dímeros de Pirimidina , Proteínas Arqueais/química , Catálise , Cristalografia/métodos , Desoxirribodipirimidina Fotoliase/química , DNA/química , DNA/efeitos da radiação , Methanosarcina/enzimologia , Conformação Proteica , Dímeros de Pirimidina/química , Raios UltravioletaRESUMO
Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FADâ¢- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FADâ¢-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.
Assuntos
Desoxirribodipirimidina Fotoliase , Prótons , Arginina/metabolismo , Cristalografia , Desoxirribodipirimidina Fotoliase/química , Desoxirribodipirimidina Fotoliase/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Transporte de Elétrons , Elétrons , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas , OxirreduçãoRESUMO
It was reported in 1995 that T7 and Taq DNA polymerases possess 3'-esterase activity, but without follow-up studies. Here we report that the 3'-esterase activity is intrinsic to the Thermococcus sp. 9°N DNA polymerase, and that it can be developed into a continuous method for DNA sequencing with dNTP analogs carrying a 3'-ester with a fluorophore. We first show that 3'-esterified dNTP can be incorporated into a template-primer DNA, and solve the crystal structures of the reaction intermediates and products. Then we show that the reaction can occur continuously, modulated by active site residues Tyr409 and Asp542. Finally, we use 5'-FAM-labeled primer and esterified dNTP with a dye to show that the reaction can proceed to ca. 450 base pairs, and that the intermediates of many individual steps can be identified. The results demonstrate the feasibility of a 3'-editing based DNA sequencing method that could find practical applications after further optimization.
Assuntos
Proteínas Arqueais/química , Carboxilesterase/química , DNA Polimerase Dirigida por DNA/química , Análise de Sequência de DNA/métodos , Thermococcus/enzimologia , Proteínas Arqueais/metabolismo , Carboxilesterase/metabolismo , DNA/química , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli , Cinética , Modelos Moleculares , Thermococcus/químicaRESUMO
Cyclobutane pyrimidine dimer (CPD) photolyases harness the energy of blue light to repair UV-induced DNA CPDs. Upon binding, CPD photolyases cause the photodamage to flip out of the duplex DNA and into the catalytic site of the enzyme. This process, called base-flipping, induces a kink in the DNA, as well as an unpaired bubble, which are stabilized by a network of protein-nucleic acid interactions. Previously, several co-crystal structures have been reported in which the binding mode of CPD photolyases has been studied in detail. However, in all cases the internucleoside linkage of the photodamage site was a chemically synthesized formacetal analogue and not the natural phosphodiester. Here, the first crystal structure and conformational analysis via molecular-dynamics simulations of a class II CPD photolyase in complex with photodamaged DNA that contains a natural cyclobutane pyrimidine dimer with an intra-lesion phosphodiester linkage are presented. It is concluded that a highly conserved bubble-intruding region (BIR) mediates stabilization of the open form of CPD DNA when complexed with class II CPD photolyases.
RESUMO
Cell-cell and cell-substrate based adhesion of yeasts are major determinants of their adoption of different life styles. Genome-mining of ascomycetous GPI-anchored cell wall proteins with lectin-like PA14 domains identified a unique class of putative adhesins in the clade of methylotrophic Komagataella yeasts, many of which are known to colonize plants and insects involving yet unknown adhesion mechanisms. Here, we report the functional and structural analysis of two of its members: KpFlo1 (=Cea1), that is highly specific for terminal N-acetylglucosamine moieties, and KpFlo2, which represents an orphan lectin with intact binding site but unknown specificity. Crystal structures of the Cea1 adhesion domain complexed to N-acetylglucosamine and N,N'-diacetylchitobiose reveal a Ca2+-dependent binding mode that differs from other members of the PA14/Flo5 adhesin family. Heterologous expression of Cea1A in Saccharomyces cerevisiae promotes cellular adhesion to non-reducing ends of non-crystalline chitin. Overall, our data suggest that high-affinity recognition of ß-GlcNAc-capped glycans by Cea1 enable Komagataella species to interact with surface cues present in fungi and insects.
RESUMO
The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins < or =40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Helicobacter pylori/genética , Proteoma , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Clonagem Molecular , Cristalização , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Helicobacter pylori/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Although the mechanisms underlying selective targeting of tail-anchored (TA) membrane proteins are well established in mammalian and yeast cells, little is known about their role in mediating intracellular membrane trafficking in plant cells. However, a recent study suggested that, in green algae, arsenite transporters located in the cytosol (ArsA1 and ArsA2) control the insertion of TA proteins into the membrane-bound organelles. In the present work, we overproduced and purified these hydrophilic proteins to near homogeneity. The analysis of their catalytic properties clearly demonstrates that C. reinhardtii ArsA proteins exhibit oxyanion-independent ATPase activity, as neither arsenite nor antimonite showed strong effects. Co-expression of ArsA proteins with TA-transmembrane regions showed not only that the former interact with the latter, but that ArsA1 does not share the same ligand specificity as ArsA2. Together with a structural model and molecular dynamics simulations, we propose that C. reinhadtii ArsA proteins are not arsenite transporters, but a TA-protein targeting factor. Further, we propose that ArsA targeting specificity is achieved at the ligand level, with ArsA1 mainly carrying TA-proteins to the chloroplast, while ArsA2 to the endoplasmic reticulum.
Assuntos
Arsenitos/metabolismo , Chlamydomonas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , ATPases Transportadoras de Arsenito/metabolismo , Modelos Moleculares , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O2 to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key ß-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn75, Asn238, and Asn458 were elucidated. Initial rate kinetic analysis revealed that the kcat, Km, and kcat/Km of nLcc4 with substrate ABTS were 3,382 s-1, 65.0 ± 6.5 µM, and 52 s-1µM-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s-1, 56.7 ± 3.2 µM, and 0.004 s-1µM-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.