RESUMEN

The histidine ATP-binding cassette (ABC) transporter of Salmonella enterica serovar Typhimurium is among the best-studied type I ABC import systems. The transporter consists of two transmembrane subunits, HisQ and HisM, and a homodimer of the nucleotide-binding subunit, HisP. Substrates are delivered by two periplasmic solute binding proteins, HisJ and LAO, with preferences for histidine and for lysine, arginine, and ornithine, respectively. A homology model was built by using the arginine-bound crystal structure of the closely related Art(QN)2 transporter of Thermoanaerobacter tengcongensis as the template. In the homodimeric Art(QN)2, one substrate molecule is bound to each of the ArtQ subunits, whereas the structural model and sequence alignments predict only one substrate molecule in contact with HisM. To address the question whether one or two binding sites exist in heterodimeric HisQM, we have studied the functional consequences of mutations by monitoring (i) the complementation of growth on d-histidine of auxotrophic tester strains, (ii) the growth of tester strains on arginine as a nitrogen source, and (iii) ATPase activity of purified variants in a lipid environment. Our results demonstrate that two negatively charged residues, namely, HisM-E166 and HisQ-D61, are indispensable for function. Furthermore, the complete reconstruction of an ArtQ-like binding site in HisQ resulted in an inactive transporter. Likewise, switching the positions of both negatively charged residues between HisQ and HisM caused transport-deficient phenotypes. Thus, we propose that one substrate molecule is primarily liganded by residues of HisM while HisQ-D61 forms a crucial salt bridge with the α-amino group of the substrate.IMPORTANCE Canonical ATP-binding cassette (ABC) importers are major players in the translocation of numerous nutrients, vitamins, and growth factors to the cytoplasm of prokaryotes. Moreover, some ABC importers have been identified as virulence factors in bacterial pathogenesis. Thus, a full understanding of their mode of action is considered a prerequisite, among others, for the development of novel antibacterial drugs. However, mainly owing to the lack of structural information, the knowledge of the chemical nature and number of substrate binding sites formed by the transmembrane subunits of ABC importers is scarce. Here, we provide evidence from mutational analyses that, in contrast to homologous homodimeric systems, the heterodimeric histidine transporter of Salmonella enterica serovar Typhimurium is liganding only one substrate molecule between its transmembrane subunits, HisM and HisQ.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Salmonella typhimurium/enzimología , Sitios de Unión , Medios de Cultivo/química , Análisis Mutacional de ADN , Mutagénesis Sitio-Dirigida , Salmonella typhimurium/genética , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismo

RESUMEN

ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN)2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243-5248, 2015, https://doi.org/10.1073/pnas.1415037112). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP)2 transporter of Geobacillus stearothermophilus, by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P]2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer.IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus, encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Aminoácidos Básicos/metabolismo , Proteínas Bacterianas/metabolismo , Geobacillus stearothermophilus/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Dimerización , Geobacillus stearothermophilus/química , Geobacillus stearothermophilus/genética

RESUMEN

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate a variety of substrates, ranging from ions to macromolecules, either out of or into the cytosol (hence defined as importers or exporters, respectively). It has been demonstrated that ABC exporters and importers function through a common mechanism involving conformational switches between inward-facing and outward-facing states; however, the mechanism underlying their functions, particularly substrate recognition, remains elusive. Here we report the structures of an amino acid ABC importer Art(QN)2 from Thermoanaerobacter tengcongensis composed of homodimers each of the transmembrane domain ArtQ and the nucleotide-binding domain ArtN, either in its apo form or in complex with substrates (Arg, His) and/or ATPs. The structures reveal that the straddling of the TMDs around the twofold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD has a negatively charged pocket that together create a negatively charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Thermoanaerobacter/enzimología , Adenosina Trifosfato/metabolismo , Apoproteínas/metabolismo , Arginina/metabolismo , Sitios de Unión , Ligandos , Modelos Moleculares , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/química , Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas Bacterianas/química , Proteínas Portadoras/química , Histidina/química , Fragmentos de Péptidos/química , Subunidades de Proteína/química , Salmonella typhimurium/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Transporte Biológico Activo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cationes Bivalentes , Escherichia coli/genética , Escherichia coli/metabolismo , Histidina/metabolismo , Hidrólisis , Magnesio/química , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/enzimología , Homología de Secuencia de Aminoácido

RESUMEN

Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

Asunto(s)

Benzodioxoles/química , Colorantes Fluorescentes/química , Proteínas de Unión a Maltosa/metabolismo , Benzodioxoles/síntesis química , Transporte de Electrón , Colorantes Fluorescentes/síntesis química , Proteínas de Unión a Maltosa/química , Estructura Terciaria de Proteína , Teoría Cuántica

RESUMEN

The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a 'gate' by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Geobacillus stearothermophilus/química , Subunidades de Proteína/química , Proteolípidos/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/química , Salmonella typhimurium/genética , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad

RESUMEN

Lon protease is a major protease in cellular protein quality control, but also plays an important regulatory role by degrading various naturally unstable regulators. Here, we traced additional such regulators by identifying regulons with co-ordinately altered expression in a lon mutant by genome-wide transcriptional profiling. Besides many members of the RcsA regulon (which validates our approach as RcsA is a known Lon substrate), many genes of the sigmaS-dependent general stress response were upregulated in the lon mutant. However, the lon mutation did not affect sigmaS levels nor sigmaS activity in general, suggesting specific effects of Lon on secondary regulators involved in the control of subsets of sigmaS-controlled genes. Lon-affected genes also included the major acid resistance genes (gadA, gadBC, gadE, hdeAB and hdeD), which led to the discovery that the essential acid resistance regulator GadE (whose expression is sigmaS-controlled) is degraded in vivo in a Lon-dependent manner. GadE proteolysis is constitutive as it was observed even under conditions that induce the system (i.e. at low pH or during entry into stationary phase). GadE degradation was found to rapidly terminate the acid resistance response upon shift back to neutral pH and to avoid overexpression of acid resistance genes in stationary phase.

Asunto(s)

Ácidos/toxicidad , Escherichia coli/fisiología , Regulación Bacteriana de la Expresión Génica , Proteasa La/metabolismo , Adaptación Fisiológica , Fusión Artificial Génica , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Reporteros , Proteasa La/genética , Regulón , Factor sigma/metabolismo , Factores de Transcripción/metabolismo , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genética

RESUMEN

The sigmaS (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, sigmaS is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of sigmaS and that sigmaS should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a sigmaS consensus promoter in silico. Moreover, our results suggest that sigmaS-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect sigmaS and/or sigma70 selectivity of many promoters. These observations indicate that certain modules of the sigmaS-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with sigma70 RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation.

Asunto(s)

Proteínas Bacterianas/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Factor sigma/fisiología , Secuencia de Bases , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Presión Osmótica , Regiones Promotoras Genéticas/fisiología