RESUMEN
The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.
Asunto(s)
Adhesinas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Adhesinas de Escherichia coli/química , Regulación Alostérica , Escherichia coli/química , Proteínas Fimbrias/química , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.
Asunto(s)
Proteínas Quinasas/metabolismo , Shigella flexneri/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Factores de Virulencia/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Proteínas Quinasas/química , Multimerización de Proteína , Ubiquitina/química , Enzimas Ubiquitina-Conjugadoras/química , Factores de Virulencia/químicaRESUMEN
BACKGROUND: Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure. METHODS: The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures. RESULTS: Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F-G loop regions and active site cavity volumes for the F87A mutated structures. CONCLUSIONS: Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site. GENERAL SIGNIFICANCE: The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.
Asunto(s)
Bacillus megaterium/enzimología , Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , NADPH-Ferrihemoproteína Reductasa/química , Quinonas/química , Bacillus megaterium/genética , Proteínas Bacterianas/genética , Sitios de Unión , Sistema Enzimático del Citocromo P-450/genética , NADPH-Ferrihemoproteína Reductasa/genética , Fenilalanina/química , Fenilalanina/genética , Unión Proteica , Relación Estructura-Actividad , Treonina/química , Treonina/genéticaRESUMEN
We report a detailed study of a point mutation of the crucial binding site residue, D128, in the biotin-streptavidin complex. The conservative substitution, D128N, preserves the detailed structure observed for the wild-type complex but has an only minimal impact on biotin binding, even though previous experimental and computational studies suggested that a charged D128 residue was crucial for high-affinity binding. These results show clearly that the fundamental basis for streptavidin's extremely strong biotin binding affinity is more complex than assumed and illustrate some of the challenges that may arise when analyzing extremely strong ligand-protein binding interactions.
Asunto(s)
Ácido Aspártico/metabolismo , Biotina/metabolismo , Mutación , Estreptavidina/metabolismo , Sitios de Unión , Cristalografía , Estreptavidina/químicaRESUMEN
Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications. In addition, it is essential to fully understand the structure and function of invertebrate GSTs, which play important roles in basic biological processes. Invertebrates harbor delta- and epsilon-class GSTs, which are not found in other organisms. Drosophila melanogaster GSTs (DmGSTs) are likely to contribute to detoxication or antioxidative stress during development, but they have not been fully characterized. Here, the structures of two epsilon-class GSTs from Drosophila, DmGSTE6 and DmGSTE7, are reported at 2.1 and 1.5â Å resolution, respectively, and are compared with other GSTs to identify structural features that might correlate with their biological functions. The structures of DmGSTE6 and DmGSTE7 are remarkably similar; the structures do not reveal obvious sources of the minor functional differences that have been observed. The main structural difference between the epsilon- and delta-class GSTs is the longer helix (A8) at the C-termini of the epsilon-class enzymes.
Asunto(s)
Proteínas de Drosophila/química , Drosophila melanogaster/química , Glutatión Transferasa/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas de Drosophila/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estabilidad Proteica , Alineación de Secuencia , TemperaturaRESUMEN
To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior. A hexameric oligomer of the cyclic peptide is found in both crystal forms and indicates that under appropriate conditions, this minimized system may also serve as a model for protein-protein interactions.
Asunto(s)
Péptidos/química , Pliegue de Proteína , Multimerización de Proteína , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de ProteínaRESUMEN
The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1-3 (Tse1-3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2-Tsi2 effector-immunity pair has features distinguishing it from previously characterized toxin-immunity and toxin-antitoxin systems.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Multimerización de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/genéticaRESUMEN
There is increasing evidence that the catch bond mechanism, where binding becomes stronger under tensile force, is a common property among non-covalent interactions between biological molecules that are exposed to mechanical force in vivo. Here, by using the multi-protein tip complex of the mannose-binding type 1 fimbriae of Escherichia coli, we show how the entire quaternary structure of the adhesive organella is adapted to facilitate binding under mechanically dynamic conditions induced by flow. The fimbrial tip mediates shear-dependent adhesion of bacteria to uroepithelial cells and demonstrates force-enhanced interaction with mannose in single molecule force spectroscopy experiments. The mannose-binding, lectin domain of the apex-positioned adhesive protein FimH is docked to the anchoring pilin domain in a distinct hooked manner. The hooked conformation is highly stable in molecular dynamics simulations under no force conditions but permits an easy separation of the domains upon application of an external tensile force, allowing the lectin domain to switch from a low- to a high-affinity state. The conformation between the FimH pilin domain and the following FimG subunit of the tip is open and stable even when tensile force is applied, providing an extended lever arm for the hook unhinging under shear. Finally, the conformation between FimG and FimF subunits is highly flexible even in the absence of tensile force, conferring to the FimH adhesin an exploratory function and high binding rates. The fimbrial tip of type 1 Escherichia coli is optimized to have a dual functionality: flexible exploration and force sensing. Comparison to other structures suggests that this property is common in unrelated bacterial and eukaryotic adhesive complexes that must function in dynamic conditions.
Asunto(s)
Adhesinas de Escherichia coli/química , Escherichia coli/fisiología , Proteínas Fimbrias/química , Fimbrias Bacterianas/fisiología , Estrés Mecánico , Adhesión Bacteriana , Escherichia coli/química , Fimbrias Bacterianas/química , Humanos , Manosa/metabolismo , Estructura Cuaternaria de Proteína , Resistencia a la Tracción , Células Tumorales CultivadasRESUMEN
Circular permutation of streptavidin was carried out in order to investigate the role of a main-chain amide in stabilizing the high-affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N-termini at residues 49 and 50 in a flexible loop involved in stabilizing the biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild-type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide-biotin hydrogen bond. In wild-type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the ß-barrel protein. A molecular-dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin-biotin complex.
Asunto(s)
Proteínas Bacterianas/química , Biotina/análogos & derivados , Proteínas Mutantes/química , Estreptavidina/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biotina/química , Biotina/metabolismo , Rastreo Diferencial de Calorimetría , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis , Proteínas Mutantes/metabolismo , Conformación Proteica , Estreptavidina/metabolismo , Termodinámica , Difracción de Rayos XRESUMEN
We report a point mutation in the second contact shell of the high-affinity streptavidin-biotin complex that appears to reduce binding affinity through transmitted effects on equilibrium dynamics. The Y54F streptavidin mutation causes a 75-fold loss of binding affinity with 73-fold faster dissociation, a large loss of binding enthalpy (ΔΔH = 3.4 kcal/mol at 37 °C), and a small gain in binding entropy (TΔΔS = 0.7 kcal/mol). The removed Y54 hydroxyl is replaced by a water molecule in the bound structure, but there are no observable changes in structure in the first contact shell and no additional changes surrounding the mutation. Molecular dynamics simulations reveal a large increase in the atomic fluctuation amplitudes for W79, a key biotin contact residue, compared to the fluctuation amplitudes in the wild-type. The increased W79 atomic fluctuation amplitudes are caused by loss of water-mediated hydrogen bonds between the Y54 hydroxyl group and peptide backbone atoms in and near W79. We propose that the increased atomic fluctuation amplitudes diminish the integrity of the W79-biotin interaction and represents a loosening of the "tryptophan collar" that is critical to the slow dissociation and high affinity of streptavidin-biotin binding. These results illustrate how changes in protein dynamics distal to the ligand binding pocket can have a profound impact on ligand binding, even when equilibrium structure is unperturbed.
Asunto(s)
Biotina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación Puntual , Estreptavidina/química , Estreptavidina/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cinética , Simulación de Dinámica Molecular , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica , Estreptavidina/genética , TermodinámicaRESUMEN
The FimH protein of Escherichia coli is a model two-domain adhesin that is able to mediate an allosteric catch bond mechanism of bacterial cell attachment, where the mannose-binding lectin domain switches from an 'inactive' conformation with fast binding to mannose to an 'active' conformation with slow detachment from mannose. Because mechanical tensile force favors separation of the domains and, thus, FimH activation, it has been thought that the catch bonds can only be manifested in a fluidic shear-dependent mode of adhesion. Here, we used recombinant FimH variants with a weakened inter-domain interaction and show that a fast and sustained allosteric activation of FimH can also occur under static, non-shear conditions. Moreover, it appears that lectin domain conformational activation happens intrinsically at a constant rate, independently from its ability to interact with the pilin domain or mannose. However, the latter two factors control the rate of FimH deactivation. Thus, the allosteric catch bond mechanism can be a much broader phenomenon involved in both fast and strong cell-pathogen attachments under a broad range of hydrodynamic conditions. This concept that allostery can enable more effective receptor-ligand interactions is fundamentally different from the conventional wisdom that allostery provides a mechanism to turn binding off under specific conditions.
Asunto(s)
Adhesinas de Escherichia coli , Adhesión Bacteriana , Escherichia coli , Proteínas Fimbrias , Adhesinas de Escherichia coli/química , Adhesinas de Escherichia coli/genética , Adhesinas de Escherichia coli/fisiología , Regulación Alostérica , Adhesión Bacteriana/fisiología , Escherichia coli/fisiología , Proteínas Fimbrias/química , Proteínas Fimbrias/genética , Manosa/metabolismo , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resistencia al CorteRESUMEN
Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95â Å, respectively. The wild-type protein crystallized with a tetramer in the asymmetric unit, while the crystals of the biotin complex contained two subunits in the asymmetric unit. Comparison of the six subunits shows the various ways in which the protein accommodates ligand binding and different crystal-packing environments. Conformational variation is found in each of the polypeptide loops connecting the eight strands in the ß-sandwich subunit, but the largest differences are found in the flexible binding loop (residues 45-52). In three of the unliganded subunits the loop is in an `open' conformation, while in the two subunits binding biotin, as well as in one of the unliganded subunits, this loop `closes' over the biotin-binding site. The `closed' loop contributes to the protein's high affinity for biotin. Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of the protein to bind biotin so tightly.
Asunto(s)
Biotina/química , Cristalografía por Rayos X/métodos , Estreptavidina/química , Estructura Secundaria de ProteínaRESUMEN
Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity toward them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is preorganized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and an engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, cocrystallized with a GSH-HNE conjugate analogue, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead, the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity toward a toxic racemate, concomitant with high catalytic efficiency and substrate specificity toward an endogenously formed toxin.
Asunto(s)
Aldehídos/metabolismo , Aldehídos/toxicidad , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Aldehídos/química , Biocatálisis , Dominio Catalítico/genética , Cristalografía por Rayos X , Glutatión Transferasa/genética , Humanos , Ligandos , Estrés Oxidativo/genética , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidad por Sustrato/genéticaRESUMEN
We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in biotin binding affinity without disrupting the equilibrium complex structure. The F130L mutation creates a small cavity occupied by a water molecule; however, all neighboring side chain positions are preserved, and protein-biotin hydrogen bonds are unperturbed. Molecular dynamics simulations reveal a reduced mobility of biotin binding residues but no observable destabilization of protein-ligand interactions. Our combined structural and computational studies suggest that the additional water molecule may affect binding affinity through an electronic polarization effect that impacts the highly cooperative hydrogen bonding network in the biotin binding pocket.
Asunto(s)
Biotina/química , Electrones , Simulación de Dinámica Molecular , Mutación Puntual , Estreptavidina/química , Estreptavidina/metabolismo , Sitios de Unión/genética , Biotina/antagonistas & inhibidores , Biotina/metabolismo , Enlace de Hidrógeno , Leucina/química , Leucina/genética , Leucina/metabolismo , Ligandos , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Unión Proteica/genética , Estabilidad Proteica , Estreptavidina/antagonistas & inhibidores , Estreptavidina/genética , TermodinámicaRESUMEN
Fimbriae and pili are macromolecular structures on the surface of Gram negative bacteria that are important for cellular adhesion. A 2.7Å resolution crystal structure of a complex of Escherichia coli fimbrial proteins containing FimH, FimG, FimF, and FimC provides the most complete model to date for the arrangement of subunits assembled in the native structure. The first three proteins form the tip of the fimbriae while FimC is the chaperone protein involved in the usher/chaperone assembly process. The subunits interact through donor strand complementation where a ß-strand from a subunit completes the ß-sandwich structure of the neighboring subunit or domain closer to the tip of the fimbria. The function of FimC is to provide a surrogate donor strand before delivery of each subunit to the FimD usher and the growing fimbria. Comparison of the subunits in this structure and their chaperone-bound complexes show that the two FimH domains change their relative orientation and position in forming the tip structure. Also, the non-chaperone subunits undergo a conformational change in their first ß-strand when the chaperone is replaced by the native donor strand. Some residues move as much as 14Å in the process. This structural shift has not been noted in structural studies of other bacterial adhesion sub-structures assembled via donor strand complementation. The domains undergo a significant structural change in the donor strand binding groove during fimbrial assembly, and this likely plays a role in determining the specificity of subunit-subunit interactions among the fimbrial proteins.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Adhesinas de Escherichia coli/química , Adhesinas de Escherichia coli/genética , Adhesinas de Escherichia coli/metabolismo , Secuencia de Aminoácidos , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9 A resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 A) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 A in some zinc-protein and zinc-ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available.
Asunto(s)
Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/química , Inhibidores de Anhidrasa Carbónica/metabolismo , Anhidrasa Carbónica II/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The specificity of human glutathione transferase (GST) A1-1 is drastically altered to favor alkenal substrates in the GIMFhelix mutant designed to mimic first-sphere interactions utilized by GSTA4-4. This redesign serves as a model for improving our understanding of the structural determinants that contribute to the distinct specificities of alpha class GSTs. Herein we report the first crystal structures of GIMFhelix, both in complex with GSH and in apo form at 1.98 and 2.38 A resolution. In contrast to the preorganized hydrophobic binding pocket that accommodates alkenals in GSTA4-4, GSTA1-1 includes a dynamic alpha9 helix that undergoes a ligand-dependent localization to complete the active site. Comparisons of the GIMFhelix structures with previously reported structures show a striking similarity with the GSTA4-4 active site obtained within an essentially GSTA1-1 scaffold and reveal the alpha9 helix assumes a similar localized structure regardless of active site occupancy in a manner resembling that of GSTA4-4. However, we cannot fully account for all the structural elements important in GSTA4-4 within the mutant's active site. The contribution of Phe10 to the Tyr212-Phe10-Phe220 network prevents complete C-terminal closure and demonstrates that the presence of Phe10 within the context of a GSTA4-4-like active site may ultimately hinder Phe220, a key C-terminal residue, from effectively contributing to the active site. In total, these results illustrate the remaining structural differences presumably reflected in the previously reported catalytic efficiencies of GIMFhelix and GSTA4-4 and emphasize the F10P mutation as being necessary to completely accomplish the transformation to a highly specific GST from the more promiscuous GSTA1-1 enzyme.
Asunto(s)
Alquenos , Glutatión Transferasa , Isoenzimas , Estructura Terciaria de Proteína , Especificidad por Sustrato/genética , Alquenos/química , Alquenos/toxicidad , Catálisis , Dominio Catalítico/genética , Cristalografía por Rayos X , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-DirigidaRESUMEN
We present a 250 ns simulation of the wild-type, biotin-liganded streptavidin tetramer in the solution phase and compare the trajectory to two previously published simulations of the protein in its crystal lattice. By performing both types of simulations, we are able to interpret the protein's behavior in solution in the context of its X-ray structure. We find that the rate of conformational sampling is increased in solution over the lattice environment, although the relevant conformational space in solution is also much larger, as indicated by overall fluctuations in the positions of backbone atoms. We also compare the distributions of chi1 angles sampled by side chains exposed to solvent in the lattice and in the solution phase, obtaining overall good agreement between the distributions obtained in our most rigorous lattice simulation and the crystallographic chi1 angles. We observe changes in the chi1 distributions in the solution phase, and note an apparent progression of the distributions as the environment changes from a tightly packed lattice filled with crystallization media to a bath of pure water. Finally, we examine the interaction of biotin and streptavidin in each simulation, uncovering a possible alternate conformation of the biotin carboxylate tail. We also note that a hydrogen bond observed to break transiently in previous solution-phase simulations is predominantly broken in this much longer solution-phase trajectory; in the lattice simulations, the lattice environment appears to help maintain the hydrogen bond, but more sampling will be needed to confirm whether the simulation model truly gives good agreement with the X-ray data in the lattice simulations. We expect that pairing solution-phase biomolecular simulations with crystal lattice simulations will help to validate simulation models and improve the interpretation of experimentally determined structures.
Asunto(s)
Biotina/química , Biotina/metabolismo , Modelos Moleculares , Estreptavidina/química , Estreptavidina/metabolismo , Cristalografía por Rayos X , Transición de Fase , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Soluciones , Factores de TiempoRESUMEN
Gene duplication and subsequent evolutionary divergence have allowed conserved proteins to develop unique roles. The MarR family of transcription factors (TFs) has undergone extensive duplication and diversification in bacteria, where they act as environmentally responsive repressors of genes encoding efflux pumps that confer resistance to xenobiotics, including many antimicrobial agents. We have performed structural, functional, and genetic analyses of representative members of the SlyA/RovA lineage of MarR TFs, which retain some ancestral functions, including repression of their own expression and that of divergently transcribed multidrug efflux pumps, as well as allosteric inhibition by aromatic carboxylate compounds. However, SlyA and RovA have acquired the ability to countersilence horizontally acquired genes, which has greatly facilitated the evolution of Enterobacteriaceae by horizontal gene transfer. SlyA/RovA TFs in different species have independently evolved novel regulatory circuits to provide the enhanced levels of expression required for their new role. Moreover, in contrast to MarR, SlyA is not responsive to copper. These observations demonstrate the ability of TFs to acquire new functions as a result of evolutionary divergence of both cis-regulatory sequences and in trans interactions with modulatory ligands.IMPORTANCE Bacteria primarily evolve via horizontal gene transfer, acquiring new traits such as virulence and antibiotic resistance in single transfer events. However, newly acquired genes must be integrated into existing regulatory networks to allow appropriate expression in new hosts. This is accommodated in part by the opposing mechanisms of xenogeneic silencing and countersilencing. An understanding of these mechanisms is necessary to understand the relationship between gene regulation and bacterial evolution. Here we examine the functional evolution of an important lineage of countersilencers belonging to the ancient MarR family of classical transcriptional repressors. We show that although members of the SlyA lineage retain some ancestral features associated with the MarR family, their cis-regulatory sequences have evolved significantly to support their new function. Understanding the mechanistic requirements for countersilencing is critical to understanding the pathoadaptation of emerging pathogens and also has practical applications in synthetic biology.
Asunto(s)
Enterobacteriaceae/genética , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Factores de Transcripción/genética , Transferencia de Gen HorizontalRESUMEN
A 250 ns molecular dynamics simulation of the biotin-liganded streptavidin crystal lattice, including cryoprotectant molecules and crystallization salts, is compared to a 250 ns simulation of the lattice solvated with pure water. The simulation using detailed crystallization conditions preserves the initial X-ray structure better than the simulation using pure water, even though the protein molecules display comparable mobility in either simulation. Atomic fluctuations computed from the simulation with crystallization conditions closely reproduce fluctuations derived from experimental temperature factors (correlation coefficient of 0.88, omitting two N-terminal residues with very high experimental B-factors). In contrast, fluctuations calculated from the simulation with pure water were less accurate, particularly for two of the streptavidin loops exposed to solvent in the crystal lattice. Finally, we obtain good agreement between the water and cryoprotectant densities obtained from the simulated crystallization conditions and the electron density due to solvent molecules in the X-ray structure. Our results suggest that detailed lattice simulations with realistic crystallization conditions can be used to assess potential function parameters, validate simulation protocols, and obtain valuable insights that solution-phase simulations do not easily provide. We anticipate that this will prove to be a powerful strategy for molecular dynamics simulations of biomolecules.