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PURPOSE: Correction of cleft nose deformity in patients with unilateral cleft lip is challenging and involves primarily the nasal cartilage and the nasal entrance. No consensus on the most effective surgical technique has been reached. This article describes a surgical procedure for nasal entrance correction. PATIENTS AND METHODS: In this retrospective study, 30 adult patients underwent secondary nasal entrance corrections. According to a modified Van der Meulen technique, a nasal alar rim flap with anatomic repositioning of the alar cartilage was applied. Symmetry and esthetic results were evaluated by semiquantitative photographic analysis. RESULTS: In all patients, the nasal tip was narrowed considerably, and a lifting of the nasal tip was achieved. Columellar elongation averaged 40%, and the form of the nostril was changed from horizontally oval to longitudinally oval. CONCLUSION: The described technique is well suited for a sustainable correction of complex cleft-induced deformities without visible scars in adult patients.
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Labio Leporino/cirugía , Cavidad Nasal/cirugía , Procedimientos Quirúrgicos Operativos/métodos , Adulto , Femenino , Humanos , Masculino , Estudios RetrospectivosRESUMEN
Interference with protein-protein interactions of interfaces larger than 1500 Ų by small drug-like molecules is notoriously difficult, particularly if targeting homodimers. The tRNA modifying enzyme Tgt is only functionally active as a homodimer. Thus, blocking Tgt dimerization is a promising strategy for drug therapy as this protein is key to the development of Shigellosis. Our goal was to identify hot-spot residues which, upon mutation, result in a predominantly monomeric state of Tgt. The detailed understanding of the spatial location and stability contribution of the individual interaction hot-spot residues and the plasticity of motifs involved in the interface formation is a crucial prerequisite for the rational identification of drug-like inhibitors addressing the respective dimerization interface. Using computational analyses, we identified hot-spot residues that contribute particularly to dimer stability: a cluster of hydrophobic and aromatic residues as well as several salt bridges. This in silico prediction led to the identification of a promising double mutant, which was validated experimentally. Native nano-ESI mass spectrometry showed that the dimerization of the suggested mutant is largely prevented resulting in a predominantly monomeric state. Crystal structure analysis and enzyme kinetics of the mutant variant further support the evidence for enhanced monomerization and provide first insights into the structural consequences of the dimer destabilization.
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Modelos Moleculares , Proteínas Mutantes/química , Pentosiltransferasa/química , ARN de Transferencia/metabolismo , Sustitución de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biología Computacional , Bases de Datos de Proteínas , Dimerización , Estabilidad de Enzimas , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Sistemas Especialistas , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.
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Acilcoenzima A , Proteínas Bacterianas , Dominio Catalítico , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzimología , Cristalografía por Rayos X , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Acilcoenzima A/metabolismo , Acilcoenzima A/química , Especificidad por Sustrato , Sitios de Unión , Modelos Moleculares , Enoil-CoA Hidratasa/metabolismo , Enoil-CoA Hidratasa/química , Unión Proteica , 3-Hidroxiacil-CoA Deshidrogenasas/química , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismoRESUMEN
To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.
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COVID-19 , SARS-CoV-2 , Humanos , Dominio Catalítico , Proteasas 3C de Coronavirus , Cristalografía por Rayos XRESUMEN
We identified the first small-molecule protein-protein interaction inhibitors of RUNX1/ETO tetramerization applying structure-based virtual screening guided by predicted hot spots and pockets in the interface. A 3D similarity screening revealed specific hot spot mimetics, one of which prevents the proliferation of RUNX1/ETO-dependent SKNO-1 cells at low micromolar concentration. Using solely a protein-protein complex structure to start with, this strategy can be the first step in any comparable structure-based endeavor to identify protein-protein interaction inhibitors.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Leucemia Mieloide/metabolismo , Terapia Molecular Dirigida , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/química , Línea Celular Tumoral , Proliferación Celular , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Proteína 1 Compañera de Translocación de RUNX1RESUMEN
The conversion of hits to leads in drug discovery involves the elaboration of chemical core structures to increase their potency. In fragment-based drug discovery, low-molecular-weight compounds are tested for protein binding and are subsequently modified, with the tacit assumption that the binding mode of the original hit will be conserved among the derivatives. However, deviations from binding mode conservation are rather frequently observed, but potential causes of these alterations remain incompletely understood. Here, two crystal forms of the spliceosomal RNA helicase BRR2 were employed as a test case to explore the consequences of conformational changes in the target protein on the binding behaviour of fragment derivatives. The initial fragment, sulfaguanidine, bound at the interface between the two helicase cassettes of BRR2 in one crystal form. Second-generation compounds devised by structure-guided docking were probed for their binding to BRR2 in a second crystal form, in which the original fragment-binding site was altered due to a conformational change. While some of the second-generation compounds retained binding to parts of the original site, others changed to different binding pockets of the protein. A structural bioinformatics analysis revealed that the fragment-binding sites correspond to predicted binding hot spots, which strongly depend on the protein conformation. This case study offers an example of extensive binding-mode changes during hit derivatization, which are likely to occur as a consequence of multiple binding hot spots, some of which are sensitive to the flexibility of the protein.
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ARN Helicasas , Empalmosomas , ARN Helicasas/química , ARN Helicasas/genética , ARN Helicasas/metabolismo , Ligandos , Empalmosomas/genética , Empalmosomas/metabolismo , ADN Helicasas/metabolismo , Conformación Proteica , Sitios de Unión , Unión ProteicaAsunto(s)
Antineoplásicos/farmacología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Multimerización de Proteína/efectos de los fármacos , Proteína 1 Compañera de Translocación de RUNX1/antagonistas & inhibidores , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Ratones , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Protein-protein interfaces are considered difficult targets for small-molecule protein-protein interaction modulators (PPIMs ). Here, we present for the first time a computational strategy that simultaneously considers aspects of energetics and plasticity in the context of PPIM binding to a protein interface. The strategy aims at identifying the determinants of small-molecule binding, hot spots, and transient pockets, in a protein-protein interface in order to make use of this knowledge for predicting binding modes of and ranking PPIMs with respect to their affinity. When applied to interleukin-2 (IL-2), the computationally inexpensive constrained geometric simulation method FRODA outperforms molecular dynamics simulations in sampling hydrophobic transient pockets. We introduce the PPIAnalyzer approach for identifying transient pockets on the basis of geometrical criteria only. A sequence of docking to identified transient pockets, starting structure selection based on hot spot information, RMSD clustering and intermolecular docking energies, and MM-PBSA calculations allows one to enrich IL-2 PPIMs from a set of decoys and to discriminate between subgroups of IL-2 PPIMs with low and high affinity. Our strategy will be applicable in a prospective manner where nothing else than a protein-protein complex structure is known; hence, it can well be the first step in a structure-based endeavor to identify PPIMs.
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Interleucina-2/química , Receptores de Interleucina-2/química , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos , Sitios de Unión , Bases de Datos de Proteínas , Interacciones Hidrofóbicas e Hidrofílicas , Interleucina-2/antagonistas & inhibidores , Modelos Moleculares , Unión Proteica , Receptores de Interleucina-2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , TermodinámicaRESUMEN
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15â Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.
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Proteínas , Ácido Aspártico Endopeptidasas , Cristalografía por Rayos X , Ligandos , Sustancias Macromoleculares , Proteínas/química , TemperaturaRESUMEN
Reduction in jaw opening is a neglected symptom of giant cell arteritis (GCA) in clinical practice and in the scientific literature. We describe the case of a 71-year-old woman with GCA who was misdiagnosed as occipital neuritis and craniomandibular dysfunction because of headaches in the occipital region and reduction in jaw opening. The reported case reminds us not to overlook reduction in jaw opening as a symptom to reveal GCA in elderly patients.
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Errores Diagnósticos , Arteritis de Células Gigantes/diagnóstico , Cefalea/diagnóstico , Maxilares/fisiopatología , Trismo/diagnóstico , Anciano , Trastornos Craneomandibulares/diagnóstico , Femenino , Arteritis de Células Gigantes/complicaciones , Arteritis de Células Gigantes/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Cefalea/tratamiento farmacológico , Cefalea/etiología , Humanos , Neuritis/diagnóstico , Lóbulo Occipital/patología , Prednisolona/uso terapéutico , Arterias Temporales/diagnóstico por imagen , Arterias Temporales/patología , Resultado del Tratamiento , Trismo/tratamiento farmacológico , Trismo/etiología , UltrasonografíaRESUMEN
Interference with protein-protein interfaces represents an attractive as well as challenging option for therapeutic intervention and drug design. The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, is only functional as a homodimer. Although we previously produced monomeric variants by site-directed mutagenesis, we only crystallized the functional dimer, simply because upon crystallization the local protein concentration increases and favors formation of the dimer interface, which represents an optimal and highly stable packing of the protein in the solid state. Unfortunately, this prevents access to structural information about the interface geometry in its monomeric state and complicates the development of modulators that can interfere with and prevent dimer formation. Here, we report on a cysteine-containing protein variant in which, under oxidizing conditions, a disulfide linkage is formed. This reinforces a novel packing geometry of the enzyme. In this captured quasi-monomeric state, the monomer units arrange in a completely different way and, thus, expose a loop-helix motif, originally embedded into the old interface, now to the surface. The motif adopts a geometry incompatible with the original dimer formation. Via the soaking of fragments into the crystals, we identified several hits accommodating a cryptic binding site next to the loop-helix motif and modulated its structural features. Our study demonstrates the druggability of the interface by breaking up the homodimeric protein using an introduced disulfide cross-link. By rational concepts, we increased the potency of these fragments to a level where we confirmed their binding by NMR to a nondisulfide-linked TGT variant. The idea of intermediately introducing a disulfide linkage may serve as a general concept of how to transform a homodimer interface into a quasi-monomeric state and give access to essential structural and design information.
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Disulfuros/química , Pentosiltransferasa/química , Bibliotecas de Moléculas Pequeñas/farmacología , Zymomonas/enzimología , Sitios de Unión/efectos de los fármacos , Ligandos , Modelos Moleculares , Multimerización de Proteína/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Zymomonas/químicaRESUMEN
In recent years, crystallographic fragment screening has matured into an almost routine experiment at several modern synchrotron sites. The hits of the screening experiment, i.e. small molecules or fragments binding to the target protein, are revealed along with their 3D structural information. Therefore, they can serve as useful starting points for further structure-based hit-to-lead development. However, the progression of fragment hits to tool compounds or even leads is often hampered by a lack of chemical feasibility. As an attractive alternative, compound analogs that embed the fragment hit structurally may be obtained from commercial catalogs. Here, a workflow is reported based on filtering and assessing such potential follow-up compounds by template docking. This means that the crystallographic binding pose was integrated into the docking calculations as a central starting parameter. Subsequently, the candidates are scored on their interactions within the binding pocket. In an initial proof-of-concept study using five starting fragments known to bind to the aspartic protease endothiapepsin, 28 follow-up compounds were selected using the designed workflow and their binding was assessed by crystallography. Ten of these compounds bound to the active site and five of them showed significantly increased affinity in isothermal titration calorimetry of up to single-digit micromolar affinity. Taken together, this strategy is capable of efficiently evolving the initial fragment hits without major synthesis efforts and with full control by X-ray crystallography.
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Ácido Aspártico Endopeptidasas , Cristalografía por Rayos X/métodos , Descubrimiento de Drogas/métodos , Ligandos , Modelos Moleculares , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Dominio Catalítico , Unión ProteicaRESUMEN
Muscle-invasive bladder cancer (MIBC) frequently harbors mutations in the CDKN1A gene, which encodes the tumor suppressor protein p21, with the majority of alterations truncating the peptide. The effect of these mutations is poorly understood. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell cycle and efficiently repair DNA damage, thus proceeding down the apoptotic pathway. We used synthetic CRISPR guide RNAs to ablate the whole peptide (sg12, targeting the 12th amino acid) or the C-terminal proliferating cell nuclear antigen (PCNA)-binding domain (sg109) to mimic different p21-truncating mutations compared with a negative control (sgGFP) in bladder cancer cell lines. Loss of detectable p21 and a stable truncated p21 peptide were identified in sg12 and sg109 single-cell clones, respectively. We found that p21-deficient cells (sg12) were sensitized to cisplatin, while cells harboring distally truncated p21 (sg12 clones) demonstrated enhanced cisplatin resistance. p21-deficient sg12 clones demonstrated less repair of DNA-platinum adducts and increased γ-H2AX foci after cisplatin exposure, suggesting there was persistent DNA damage after p21 loss. p21-deficient sg12 clones were also unable to prevent the activation of CDK1 after DNA damage, and therefore, continued through the cell cycle, resulting in replication fork collapse, potentially explaining the observed cisplatin sensitization. sg109 clones were neither unable to sequester PCNA nor localize p21 to the nucleus after DNA damage, potentially explaining the chemoresistant phenotype. Our findings suggest that different CDKN1A truncations have different and perhaps disparate biology, and that there may be a duality of effect on cisplatin sensitivity depending on mutation context. IMPLICATIONS: Some truncating CDKN1A mutations generate a retained peptide that may have neomorphic functions and affect cisplatin sensitivity in patients with bladder cancer.
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Cisplatino/uso terapéutico , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Cisplatino/farmacología , Humanos , MutaciónRESUMEN
Fragment screening is a technique that helps to identify promising starting points for ligand design. Given that crystals of the target protein are available and display reproducibly high-resolution X-ray diffraction properties, crystallography is among the most preferred methods for fragment screening because of its sensitivity. Additionally, it is the only method providing detailed 3D information of the binding mode of the fragment, which is vital for subsequent rational compound evolution. The routine use of the method depends on the availability of suitable fragment libraries, dedicated means to handle large numbers of samples, state-of-the-art synchrotron beamlines for fast diffraction measurements and largely automated solutions for the analysis of the results. Here, the complete practical workflow and the included tools on how to conduct crystallographic fragment screening (CFS) at the Helmholtz-Zentrum Berlin (HZB) are presented. Preceding this workflow, crystal soaking conditions as well as data collection strategies are optimized for reproducible crystallographic experiments. Then, typically in a one to two-day procedure, a 96-membered CFS-focused library provided as dried ready-to-use plates is employed to soak 192 crystals, which are then flash-cooled individually. The final diffraction experiments can be performed within one day at the robot-mounting supported beamlines BL14.1 and BL14.2 at the BESSY II electron storage ring operated by the HZB in Berlin-Adlershof (Germany). Processing of the crystallographic data, refinement of the protein structures, and hit identification is fast and largely automated using specialized software pipelines on dedicated servers, requiring little user input. Using the CFS workflow at the HZB enables routine screening experiments. It increases the chances for successful identification of fragment hits as starting points to develop more potent binders, useful for pharmacological or biochemical applications.
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Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Berlin , Cristalización , Recolección de Datos , Ligandos , Proteínas/química , Programas Informáticos , Sincrotrones , Flujo de TrabajoRESUMEN
Crystallographic fragment screening (CFS) provides excellent starting points for projects concerned with drug discovery or biochemical tool compound development. One of the fundamental prerequisites for effective CFS is the availability of a versatile fragment library. Here, we report on the assembly of the 1,103-compound F2X-Universal Library and its 96-compound sub-selection, the F2X-Entry Screen. Both represent the available fragment chemistry and are highly diverse in terms of their 3D-pharmacophore variations. Validation of the F2X-Entry Screen in CFS campaigns using endothiapepsin and the Aar2/RNaseH complex yielded hit rates of 30% and 21%, respectively, and revealed versatile binding sites. Dry presentation of the libraries allows CFS campaigns to be carried out with or without the co-solvent DMSO present. Most of the hits in our validation campaigns could be reproduced also in the absence of DMSO. Consequently, CFS can be carried out more efficiently and for a wider range of conditions and targets.
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Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Bases de Datos de Compuestos Químicos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
The publication contained a dating model that was based on AD/BC dates instead of years before present (YBP) dates for the three C14 AMS values. As a result, dates reported as YBP should be reported as BC. While all of the dates for the prescribed burning period are correct given that they were based on the 210Pb model, all dates reported as YBP should read BC. Specific changes to the manuscript are as follows: The abstract should read, "throughout the last 8000 years." The final paragraph in the introduction should read, "from the mid-Holocene (~ 6000 BC) to present." The end of the first paragraph in the Results section should read, "the sediment core represented the past ~ 8000 YBP and the core sections below the 210Pb record averaged sedimentation rates of 1.73 ± 2.1 mg cm-2 year-1." All dates presented as Years Before Present or YPB for the remainder of the manuscript should be reported as BC. Added to Acknowledgements: "The authors would like to thank Sally Horn and Matt Boehm with help with the age model and dating." Table 1 has been updated with the "Calibrated Age" column reflecting the correct dates in YBP notation. Figure 2 has been updated to reflect the BC to YBP changes in the calibrated AMS C14 dates. Both panels have been changed to include the older dates.
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Prescribed fire is a common management practice for forests and other terrestrial environments. Following a prescribed burn, ash erodes into aquatic environments potentially altering terrestrial-aquatic connectivity and water quality. In this study, we collected a sediment core from Ocean Pond, FL, USA, a lake that has received fire ash from decades of prescribed burning events. Paleolimnological measurements of macrocharcoal, nutrients, stable isotopes (δ13C, δ15N), and photosynthetic pigments were used to reconstruct fire regimes, material inputs, and lake primary producer responses for periods of prescribed burns and other lake periods throughout the last 8000 years [corrected]. Results show that the period of repeated modern-prescribed fires coincided with decreased C and N depositions in the lake, while P deposition increased causing alterations to nutrient storage and stoichiometry. However, photosynthetic pigments indicated low primary producer abundance during the prescribed fire period. These changes in nutrient dynamics could provide new insights into biogeochemical pathways in land-water connected systems where burning has not been considered.
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Ecosistema , Incendios , Bosques , Lagos , NutrientesRESUMEN
The incorporation of diamondoid amino acids (DAAs) into peptide-like drugs is a general strategy to improve lipophilicity, membrane permeability, and metabolic stability of peptidomimetic pharmaceuticals. We designed and synthesized five novel peptidic DAA-containing kinase inhibitors of protein kinaseâ A using a sophisticated molecular dynamics protocol and solid-phase peptide synthesis. By means of a thermophoresis binding assay, NMR, and crystal structure analysis, we determined the influence of the DAAs on the secondary structure and binding affinity in comparison to the native protein kinase inhibitor, which is purely composed of proteinogenic amino acids. Affinity and binding pose are largely conserved. One variant showed 6.5-fold potency improvement, most likely related to its increased side chain lipophilicity. A second variant exhibited slightly decreased affinity presumably due to loss of hydrogen-bond contacts to surrounding water molecules of the first solvation shell.
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Aminoácidos/química , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Cristalografía por Rayos X , Proteínas Quinasas Dependientes de AMP Cíclico/química , Simulación de Dinámica MolecularRESUMEN
For the efficient pathogenesis of Shigella, the causative agent of bacillary dysentery, full functionality of tRNA-guanine transglycosylase (TGT) is mandatory. TGT performs post-transcriptional modifications of tRNAs in the anticodon loop taking impact on virulence development. This suggests TGT as a putative target for selective anti-shigellosis drug therapy. Since bacterial TGT is only functional as homodimer, its activity can be inhibited either by blocking its active site or by preventing dimerization. Recently, we discovered that in some crystal structures obtained by soaking the full conformational adaptation most likely induced in solution upon ligand binding is not displayed. Thus, soaked structures may be misleading and suggest irrelevant binding modes. Accordingly, we re-investigated these complexes by co-crystallization. The obtained structures revealed large conformational rearrangements not visible in the soaked complexes. They result from spatial perturbations in the ribose-34/phosphate-35 recognition pocket and, consequently, an extended loop-helix motif required to prevent access of water molecules into the dimer interface loses its geometric integrity. Thermodynamic profiles of ligand binding in solution indicate favorable entropic contributions to complex formation when large conformational adaptations in the dimer interface are involved. Native MS titration experiments reveal the extent to which the homodimer is destabilized in the presence of each inhibitor. Unexpectedly, one ligand causes a complete rearrangement of subunit packing within the homodimer, never observed in any other TGT crystal structure before. Likely, this novel twisted dimer is catalytically inactive and, therefore, suggests that stabilizing this non-productive subunit arrangement may be used as a further strategy for TGT inhibition.
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Proteínas Bacterianas/química , Modelos Moleculares , Multimerización de Proteína , ARN de Transferencia/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Pentosiltransferasa/antagonistas & inhibidores , Pentosiltransferasa/química , Pentosiltransferasa/metabolismo , Unión Proteica , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de Proteína , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Soluciones , Termodinámica , Zymomonas/enzimologíaRESUMEN
The 2-deoxy-d-ribose-5-phosphate aldolase (DERA) offers access to highly desirable building blocks for organic synthesis by catalyzing a stereoselective C-C bond formation between acetaldehyde and certain electrophilic aldehydes. DERA´s potential is particularly highlighted by the ability to catalyze sequential, highly enantioselective aldol reactions. However, its synthetic use is limited by the absence of an enantiocomplementary enzyme. Here, we introduce the concept of homologous grafting to identify stereoselectivity-determining amino acid positions in DERA. We identified such positions by structural analysis of the homologous aldolases 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG) and the enantiocomplementary enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase (KDPGal). Mutation of these positions led to a slightly inversed enantiopreference of both aldolases to the same extent. By transferring these sequence motifs onto DERA we achieved the intended change in enantioselectivity.