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Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom (Å) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S). First, we evaluated data quality on the standard benchmarking sample, rabbit muscle aldolase, using three of the most frequently used cryo-EM data collection software: SerialEM, Leginon and EPU, and found that - despite sample heterogeneity - all final reconstructions yield same overall resolutions of 2.6 Å and map quality when using either of the three software. Furthermore, comparison between Glacios-F4-S and a 300 kV cryo-TEM (Titan Krios with Falcon 4) revealed nominal resolution differences in overall reconstructions of a reconstituted human nucleosome core particle, achieving 2.8 and 2.5 Å, respectively. Finally, we performed comparative data analysis on the human RAD51 paralog complex, BCDX2, a four-protein complex of approximately 150 kilodaltons, and found that a small dataset (≤1,000 micrographs) was sufficient to generate a 3.3 Å reconstruction, with sufficient detail to resolve co-bound ligands, AMP-PNP and Mg +2 . In summary, this study provides evidence that the Glacios-F4-S operates equally well with all standard data collection software, and is sufficient to obtain high resolution structural information of novel macromolecular complexes, readily acquiring single particle data rivaling that of 300 kV cryo-TEMs.
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Overexpression of BCL-xL and BCL-2 play key roles in tumorigenesis and cancer drug resistance. Advances in PROTAC technology facilitated recent development of the first BCL-xL/BCL-2 dual degrader, 753b, a VHL-based degrader with improved potency and reduced toxicity compared to previous small molecule inhibitors. Here, we determine crystal structures of VHL/753b/BCL-xL and VHL/753b/BCL-2 ternary complexes. The two ternary complexes exhibit markedly different architectures that are accompanied by distinct networks of interactions at the VHL/753b-linker/target interfaces. The importance of these interfacial contacts is validated via functional analysis and informed subsequent rational and structure-guided design focused on the 753b linker and BCL-2/BCL-xL warhead. This results in the design of a degrader, WH244, with enhanced potency to degrade BCL-xL/BCL-2 in cells. Using biophysical assays followed by in cell activities, we are able to explain the enhanced target degradation of BCL-xL/BCL-2 in cells. Most PROTACs are empirically designed and lack structural studies, making it challenging to understand their modes of action and specificity. Our work presents a streamlined approach that combines rational design and structure-based insights backed with cell-based studies to develop effective PROTAC-based cancer therapeutics.
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Neoplasias , Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Proteína bcl-X/metabolismoRESUMEN
ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7. Here, we present cryo-EM structures of human Uba7 in complex with UBE2L6, ISG15 adenylate, and ISG15 thioester intermediate that are poised for catalysis of Uba7-UBE2L6-ISG15 thioester transfer. Our structures reveal a unique overall architecture of the complex compared to structures from the ubiquitin conjugation pathway, particularly with respect to the location of ISG15 thioester intermediate. Our structures also illuminate the molecular basis for Uba7 activities and for its exquisite specificity for ISG15 and UBE2L6. Altogether, our structural, biochemical, and human cell-based data provide significant insights into the functions of Uba7, UBE2L6, and ISG15 in cells.
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Citocinas , Enzimas Ubiquitina-Conjugadoras , Humanos , Citocinas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Microscopía por Crioelectrón , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and the HR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.
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Proteínas de Unión al ADN , Recombinación Homóloga , Complejos Multiproteicos , Humanos , Microscopía por Crioelectrón , Replicación del ADN , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Neoplasias/genética , Nucleoproteínas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Recombinasa Rad51/ultraestructura , Especificidad por SustratoRESUMEN
The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa-fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood.
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Factor Xa , Protrombina , Microscopía por Crioelectrón , Factor V , Factor Va/metabolismo , Factor Xa/metabolismo , Protrombina/metabolismo , Tromboplastina/metabolismoRESUMEN
Coagulation factor V (fV) is the precursor of fVa, which, together with fXa, Ca2+, and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. We solved the cryogenic electron microscopy (cryo-EM) structures of human fV and fVa at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly, but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding fXa, and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain that is responsible for prothrombin binding. Ordering of this region and full exposure of the fXa epitope emerge as necessary steps in the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of fV and fVa and pioneer the analysis of coagulation factors by cryo-EM.
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Microscopía por Crioelectrón , Factor Va , Factor Va/química , Factor Va/ultraestructura , Humanos , Dominios ProteicosRESUMEN
The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of 19F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na+-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*-E equilibrium and providing no evidence that free thrombin is zymogen-like.
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Flúor/química , Espectroscopía de Resonancia Magnética , Precursores de Proteínas/metabolismo , Protrombina/metabolismo , Trombina/química , Trombina/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
Enzyme prodrug therapy has the potential to remedy the lack of selectivity associated with the systemic administration of chemotherapy. However, most current systems are immunogenic and constrained to a monotherapeutic approach. We developed a new class of fusion proteins centered about the human enzyme ß-glucuronidase (ßG), capable of converting several innocuous prodrugs into chemotherapeutics. We targeted ßG to phosphatidylserine on tumor cells, tumor vasculature and metastases via annexin A1/A5. Phosphatidylserine shows promise as a universal marker for solid tumors and allows for tumor type-independent targeting. To create fusion proteins, human annexin A1/A5 was genetically fused to the activity-enhancing 16a3 mutant of human ßG, expressed in chemically defined, fed-batch suspension culture, and chromatographically purified. All fusion constructs achieved >95% purity with yields up to 740 µg/l. Fusion proteins displayed cancer selective cell-surface binding with cell line-dependent binding stability. One fusion protein in combination with the prodrug SN-38 glucuronide was as effective as the drug SN-38 on Panc-1 pancreatic cancer cells and HAAE-1 endothelial cells, and demonstrated efficacy against MCF-7 breast cancer cells. ßG fusion proteins effectively enable localized combination therapy that can be tailored to each patient via prodrug selection, with promising clinical potential based on their near fully human design.
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Anexina A1/genética , Anexina A5/genética , Glucuronidasa/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Línea Celular Tumoral , Glucuronidasa/química , Glucuronidasa/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Terapia Molecular Dirigida , Mutación , Profármacos/metabolismo , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Caseinolytic protease P (ClpP) maintains essential roles in bacterial homeostasis. As such, both the inhibition and activation of this enzyme result in bactericidal activity, making ClpP a promising target for antibacterial drug development. Herein, we report the results of a fluorescence-based screen of â¼450 structurally diverse fungal and bacterial secondary metabolites. Sclerotiamide (1), a paraherquamide-related indolinone, was identified as the first non-peptide-based natural product activator of ClpP. Structure-activity relationships arising from the initial screen, preliminary biochemical evaluation of 1, and rationale for the exploitation of this chemotype to develop novel ClpP activators are presented.
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Productos Biológicos/química , Productos Biológicos/farmacología , Endopeptidasas/metabolismo , Indolizinas/química , Indolizinas/farmacología , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Antibacterianos/farmacología , Catálisis , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Relación Estructura-ActividadRESUMEN
OBJECTIVE: Using TCGA database, we had demonstrated that aberrantly activated Forkhead box M1 (FOXM1) correlates to worse overall survival in a subgroup of platinum resistant patients. Application of thiostrepton, a natural thiazole antibiotics that inhibits FOXM1 transcription activity in the clinic is hampered by difficulties in synthesis, degradation potential, and solubility. In this study, we aim to identify potential FOXM1 small molecule inhibitors to develop a new class of therapeutic agents to address the challenges in treating chemotherapy resistant EOC. METHODS: We used in silico screening of compounds against a solved structure of FOXM1 and subsequently to derive a list of possible compounds that could inhibit FOXM1. Three compounds were tested for in vitro cytotoxicity and FOXM1 expression level was confirmed by RT-PCR and Western blot in EOC cell lines. RESULTS: The FOXM1 structure obtained from 3G73 represented the DNA binding region of FOXM1 and possessed the winged helix fold representative of the Forkhead family of enzymes with two wings in direct contact with DNA. For ease of representation, we described both wings as a dimer and a single wing as a monomer. From this structure, we hypothesized two main models of how thiostrepton binding to FOXM1 could possibly curtail its transcriptional activity. In the first model thiostrepton could bind either of the wings or both wings and prevent association to DNA. In the second model thiostrepton bind the FOXM1/DNA complex and weaken association of FOXM1 to DNA. Subsequently, small molecular inhibitors could also use either of the models to inhibit transcription. To account for both models, the NCI diversity set was screened against the FOXM1 dimer:DNA complex (39 hits), dimer (11 hits) and monomer (14 hits). Those hits were further classified by chemical structure, biological function and chemical similarities to known molecules that target FOXM1. In cellular cytotoxicity assays, N-phenylphenanthren-9-amine (related to hit #225) successfully showed cytotoxicity to all three cell lines with IC50 around 1µM, and downregulate FOXM1 and transcription of its downstream molecules such as CCNB1. CONCLUSION: By a combination of in silico screening coupled to cellular cytotoxicity studies, we have taken the first step towards identifying potential inhibitors of FOXM1 that can replace thiostrepton.
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Factores de Transcripción Forkhead/metabolismo , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Ováricas/metabolismo , Carcinoma Epitelial de Ovario , Simulación por Computador , Femenino , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Humanos , Unión ProteicaRESUMEN
Hydrogen bonds profoundly influence the architecture and activity of biological macromolecules. Deep appreciation of hydrogen bond contributions to biomolecular function thus requires a detailed understanding of hydrogen bond structure and energetics and the relationship between these properties. Hydrogen bond formation energies (ΔGf) are enormously more favorable in aprotic solvents than in water, and two classes of contributing factors have been proposed to explain this energetic difference, focusing respectively on the isolated and hydrogen-bonded species: (I) water stabilizes the dissociated donor and acceptor groups much better than aprotic solvents, thereby reducing the driving force for hydrogen bond formation; and (II) water lengthens hydrogen bonds compared to aprotic environments, thereby decreasing the potential energy within the hydrogen bond. Each model has been proposed to provide a dominant contribution to ΔGf, but incisive tests that distinguish the importance of these contributions are lacking. Here we directly test the structural basis of model II. Neutron crystallography, NMR spectroscopy, and quantum mechanical calculations demonstrate that O-H···O hydrogen bonds in crystals, chloroform, acetone, and water have nearly identical lengths and very similar potential energy surfaces despite ΔGf differences >8 kcal/mol across these solvents. These results rule out a substantial contribution from solvent-dependent differences in hydrogen bond structure and potential energy after association (model II) and thus support the conclusion that differences in hydrogen bond ΔGf are predominantly determined by solvent interactions with the dissociated groups (model I). These findings advance our understanding of universal hydrogen-bonding interactions and have important implications for biology and engineering.
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Agua/química , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Estructura Molecular , Teoría Cuántica , Solventes/química , TermodinámicaRESUMEN
We compared the binding affinities of ground state analogues for bacterial ketosteroid isomerase (KSI) with a wild-type anionic Asp general base and with uncharged Asn and Ala in the general base position to provide a measure of potential ground state destabilization that could arise from the close juxtaposition of the anionic Asp and hydrophobic steroid in the reaction's Michaelis complex. The analogue binding affinity increased ~1 order of magnitude for the Asp38Asn mutation and ~2 orders of magnitude for the Asp38Ala mutation, relative to the affinity with Asp38, for KSI from two sources. The increased level of binding suggests that the abutment of a charged general base and a hydrophobic steroid is modestly destabilizing, relative to a standard state in water, and that this destabilization is relieved in the transition state and intermediate in which the charge on the general base has been neutralized because of proton abstraction. Stronger binding also arose from mutation of Pro39, the residue adjacent to the Asp general base, consistent with an ability of the Asp general base to now reorient to avoid the destabilizing interaction. Consistent with this model, the Pro mutants reduced or eliminated the increased level of binding upon replacement of Asp38 with Asn or Ala. These results, supported by additional structural observations, suggest that ground state destabilization from the negatively charged Asp38 general base provides a modest contribution to KSI catalysis. They also provide a clear illustration of the well-recognized concept that enzymes evolve for catalytic function and not, in general, to maximize ground state binding. This ground state destabilization mechanism may be common to the many enzymes with anionic side chains that deprotonate carbon acids.
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Alanina/metabolismo , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Comamonas testosteroni/enzimología , Pseudomonas putida/enzimología , Esteroide Isomerasas/química , Alanina/química , Alanina/genética , Asparagina/química , Asparagina/genética , Ácido Aspártico/química , Ácido Aspártico/genética , Sitios de Unión , Catálisis , Dominio Catalítico , Comamonas testosteroni/genética , Cristalografía por Rayos X , Enlace de Hidrógeno , Cetosteroides/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación/genética , Pseudomonas putida/genética , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismoRESUMEN
Enzymes are classically proposed to accelerate reactions by binding substrates within active-site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1-1 A scale of most substrate rearrangements. The flexibility of active-site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together 1.2-1.5 A resolution X-ray crystallography, (1)H and (19)F NMR spectroscopy, quantum mechanical calculations, and transition-state analogue binding measurements to test the distance scale on which noncovalent forces can constrain the structural relaxation or translation of side chains and ligands along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side-chain reorientation and prevent hydrogen bond shortening by 0.1 A or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function.
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Oxígeno/química , Esteroide Isomerasas/química , Esteroide Isomerasas/metabolismo , Aniones/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Unión Proteica , Protones , Pseudomonas putida/enzimología , Electricidad EstáticaRESUMEN
A natural bonding orbital (NBO) analysis of phosphate bonding and connection to experimental phosphotransfer potential is presented. Density functional calculations with the 6-311++G(d,p) basis set carried out on 10 model phosphoryl compounds verify that the wide variability of experimental standard free energies of hydrolysis (a phosphotransfer potential benchmark) is correlated with the instability of the scissile O-P bond through computed bond lengths. NBO analysis is used to analyze all delocalization interactions contributing to O-P bond weakening. Phosphoryl bond lengths are found to correlate strongest (R = 0.90) with the magnitude of the ground-state n(O) --> sigma*(O-P) anomeric effect. Electron-withdrawing interactions of the substituent upon the sigma(O-P) bonding orbital also correlate strongly with O-P bond lengths (R = 0.88). However, an analysis of sigma*(O-P) and sigma(O-P) populations show that the increase in sigma*(O-P) density is up to 6.5 times greater than the decrease in sigma(O-P) density. Consequently, the anomeric effect is more important than other delocalization interactions in impacting O-P bond lengths. Factors reducing anomeric power by diminishing either lone pair donor ability (solvent) or antibonding acceptor ability (substituent) are shown to result in shorter O-P bond lengths. The trends shown in this work suggest that the generalized anomeric effect provides a simple explanation for relating the sensitivity of the O-P bond to diverse environmental and substituent factors. The anomeric n(O) --> sigma*(O-P) interaction is also shown to correlate strongly with experimentally determined standard free energies of hydrolysis (R = -0.93). A causal mechanism cannot be inferred from correlation. Equally, a P-value of 1.2 x 10(-4) from an F-test indicates that it is unlikely that the ground-state anomeric effect and standard free energies of hydrolysis are coincidentally related. It is found that as the exothermicity of hydrolysis increases, the energy stabilization of the ground-state anomeric effect increases with selective destabilization of the high-energy O-P bond to be broken in hydrolysis. The anomeric effect therefore partially counteracts a larger resonance stabilization of products that makes hydrolysis exothermic and needs to be considered in achieving improved agreement between calculated and empirical energies of hydrolysis. The avenues relating the thermodynamic behavior of phosphates to underlying structural factors via the anomeric effect are discussed.
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Modelos Químicos , Fosfatos/química , Teoría Cuántica , Hidrólisis , Estructura Molecular , Electricidad Estática , TermodinámicaRESUMEN
Electronic structure calculations have been carried out to provide a molecular interpretation for dihydrogen phosphate stability in water relative to that of metaphosphate. Specifically, hydration enthalpies of biologically important metaphosphate and dihydrogen phosphate with one to three waters have been computed with second-order Møller-Plesset perturbation and density functional theory (B3LYP) with up to the aug-cc-pvtz basis set and compared to experiment. The inclusion of basis set superposition error corrections and supplemental diffuse functions are necessary to predict hydration enthalpies within experimental uncertainty. Natural bond orbital analysis is used to rationalize underlying hydrogen bond configurations and key orbital interactions responsible for the experimentally reported difference in hydration enthalpies between metaphosphate and dihydrogen phosphate. In general, dihydrogen phosphate forms stronger hydrogen bonds compared to metaphosphate due to a greater charge transfer or enhanced orbital overlap between the phosphoryl oxygen lone pairs, n(O), and the antibonding O-H bond of water. Intramolecular distal lone pair repulsion with the donor n(O) orbital of dihydrogen phosphate distorts symmetric conformations, which improves n(O) and sigma*(O-H) overlap and ultimately the hydrogen bond strength. Unlike metaphosphate, water complexed to dihydrogen phosphate can serve as both a hydrogen bond donor and a hydrogen bond acceptor, which results in cooperative charge transfer and a reduction of the energy gap between n(O) and sigma*(O-H), leading to stronger hydrogen bonds. This study offers insight into how orbital interactions mediate hydrogen bond strengths with potential implications on the understanding of the kinetics and mechanism in enzymatic phosphoryl transfer reactions.
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Algoritmos , Fosfatos/química , Agua/química , Hidrógeno/química , Enlace de Hidrógeno , Modelos Moleculares , Oxígeno/química , Ácidos Fosforosos/química , Teoría Cuántica , TermodinámicaRESUMEN
Electronic structure calculations have been performed on a model N-phosphorylguanidine, or phosphagen, to understand the stereoelectronic factors contributing to the lability of the "high-energy" N-P bond. The lability of the N-P bond is central to the physiological role of phosphagens involving phosphoryl transfer reactions important in cellular energy buffering and metabolism. Eight protonated forms of N-methyl-N'-phosphorylguanidine have been energy minimized at levels of theory ranging up to B3LYP/6-311++G(d,p) and MP2/6-311++G(d,p) to investigate the correlation between protonation state and N-P bond length. Selected forms have also been minimized using the CCSD/6-311++G(d,p) and QCISD/6-311++G(d,p) levels of theory. Bulk solvation energies using the polarized continuum model (PCM) with B3LYP/6-311++G(d,p) test the influence of the surroundings on computed structures and energies. The N-P bond length depends on the overall protonation state where increased protonation at the phosphoryl group or deprotonation at the unsubstituted N'' nitrogen results in shorter, stronger N-P bonds. Natural bond orbital analysis shows that the protonation state affects the N-P bond length by altering the magnitude of stabilizing n(O) --> sigma*(N-P) stereoelectronic interactions and to a lesser extent the sigma(N-P) --> sigma*(C-N'') and sigma(N-P) --> sigma*(C-N) interactions. The computations do not provide evidence of a competition between the phosphoryl and guanidinium groups for the same lone pair on the bridging nitrogen, as previously suggested by opposing resonance theory. The computed n(O) --> sigma*(N-P) anomeric effect provides a novel explanation of "high-energy" N-P bond lability. This offers new mechanistic insight into phosphoryl transfer reactions involving both phosphagens and other biochemically important "high-energy" phosphoester bonds.