RESUMEN
The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Descubrimiento de Drogas , ADN Polimerasa Dirigida por ADN/metabolismo , Sitios de Unión , Endonucleasas/metabolismo , Cristalografía por Rayos X , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismoRESUMEN
Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme's ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.
RESUMEN
Cancer will directly affect the lives of over one-third of the population. The DNA Damage Response (DDR) is an intricate system involving damage recognition, cell cycle regulation, DNA repair, and ultimately cell fate determination, playing a central role in cancer etiology and therapy. Two primary therapeutic approaches involving DDR targeting include: combinatorial treatments employing anticancer genotoxic agents; and synthetic lethality, exploiting a sporadic DDR defect as a mechanism for cancer-specific therapy. Whereas, many DDR proteins have proven "undruggable", Fragment- and Structure-Based Drug Discovery (FBDD, SBDD) have advanced therapeutic agent identification and development. FBDD has led to 4 (with â¼50 more drugs under preclinical and clinical development), while SBDD is estimated to have contributed to the development of >200, FDA-approved medicines. Protein X-ray crystallography-based fragment library screening, especially for elusive or "undruggable" targets, allows for simultaneous generation of hits plus details of protein-ligand interactions and binding sites (orthosteric or allosteric) that inform chemical tractability, downstream biology, and intellectual property. Using a novel high-throughput crystallography-based fragment library screening platform, we screened five diverse proteins, yielding hit rates of â¼2-8% and crystal structures from â¼1.8 to 3.2 Å. We consider current FBDD/SBDD methods and some exemplary results of efforts to design inhibitors against the DDR nucleases meiotic recombination 11 (MRE11, a.k.a., MRE11A), apurinic/apyrimidinic endonuclease 1 (APE1, a.k.a., APEX1), and flap endonuclease 1 (FEN1).
Asunto(s)
Descubrimiento de Drogas , Preparaciones Farmacéuticas , Cristalografía por Rayos X , Daño del ADN , Reparación del ADNRESUMEN
Free-standing single-layer ß-sheets are extremely rare in naturally occurring proteins, even though ß-sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single-layer, anti-parallel ß-sheet proteins, comprised of three or four twisted ß-hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of ß-sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent-exposed tyrosine residues, was identified on the concave surface of the ß-sheet. These new modular single-layer ß-sheet proteins may serve as a new model system for studying folding and design of ß-rich proteins.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/química , Bacterias/química , Cristalografía por Rayos X , Microbioma Gastrointestinal , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformación Proteica en Lámina beta , Pliegue de Proteína , Tirosina/químicaRESUMEN
Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key ß sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal ß strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.
Asunto(s)
Proteínas Fimbrias/química , Fimbrias Bacterianas , Microbioma Gastrointestinal , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein-protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39-U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35-U2AF65 and RBM39-SF3b155, the RBM39-UHM-U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM-ULM binding interface, providing a rationale for the known specificity of UHM-ULM interactions. This study therefore establishes a structural basis for specific UHM-ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.
Asunto(s)
Complejos Multiproteicos/química , Proteínas Nucleares/química , Proteínas de Unión al ARN/química , Factor de Empalme U2AF/química , Cristalografía por Rayos X , Humanos , Células Jurkat , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Estructura Cuaternaria de ProteínaRESUMEN
Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/ß/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys(147), resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys(147) to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca(2+) for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.
Asunto(s)
Proteínas Bacterianas/química , Bacteroidaceae/enzimología , Proteasas de Cisteína/química , Microbioma Gastrointestinal , Cristalografía por Rayos X , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and they likely destabilize the formation of functional complexes.
Asunto(s)
ADN/química , ADN/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Deformidades Congénitas de las Extremidades/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Cristalografía por Rayos X , Proteínas de Homeodominio/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Factores de Transcripción/genéticaRESUMEN
The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.
Asunto(s)
Fosfatidilinositoles/química , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/ultraestructura , Factor Esteroidogénico 1/ultraestructura , Sitios de Unión/fisiología , Cristalografía por Rayos X , Receptor Nuclear Huérfano DAX-1/química , Humanos , Modelos Moleculares , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Factor Esteroidogénico 1/químicaRESUMEN
UNLABELLED: Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCE: Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.
Asunto(s)
Aminopeptidasas/química , Aminopeptidasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominios Homologos src , Aminopeptidasas/genética , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalografía por Rayos X , Análisis Mutacional de ADN , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
Flavodoxins in combination with the flavin mononucleotide (FMN) cofactor play important roles for electron transport in prokaryotes. Here, novel insights into the FMN-binding mechanism to flavodoxins-4 were obtained from the NMR structures of the apo-protein from Lactobacillus acidophilus (YP_193882.1) and comparison of its complex with FMN. Extensive reversible conformational changes were observed upon FMN binding and release. The NMR structure of the FMN complex is in agreement with the crystal structure (PDB ID: 3EDO) and exhibits the characteristic flavodoxin fold, with a central five-stranded parallel ß-sheet and five α-helices forming an α/ß-sandwich architecture. The structure differs from other flavoproteins in that helix α2 is oriented perpendicular to the ß-sheet and covers the FMN-binding site. This helix reversibly unfolds upon removal of the FMN ligand, which represents a unique structural rearrangement among flavodoxins.
Asunto(s)
Mononucleótido de Flavina/química , Flavodoxina/química , Lactobacillus acidophilus/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Transporte de Electrón , Flavodoxina/metabolismo , Lactobacillus acidophilus/enzimología , Espectroscopía de Resonancia Magnética , Unión Proteica , Pliegue de ProteínaRESUMEN
NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.
Asunto(s)
Proliferación Celular/genética , Reprogramación Celular/genética , Proteínas de Homeodominio/genética , Mutación , Células Madre Pluripotentes/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Cristalografía por Rayos X , ADN/química , ADN/genética , ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Proteína Homeótica Nanog , Conformación de Ácido Nucleico , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , TransfecciónRESUMEN
GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo structure at 1.15 Šresolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predicted in silico based on structural and bioinformatics data, and subsequently were characterized experimentally. Further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines another amidase family.
Asunto(s)
Modelos Moleculares , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Peptidoglicano/metabolismo , Bacteroides , Cristalografía por Rayos X , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivKâ¼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Proteínas Quinasas/metabolismo , Ciclo Celular , Dimerización , Histidina Quinasa , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The signaling phosphatidylinositol lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP2 and PIP3 show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP3 stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As.
Asunto(s)
Fosfatidilinositoles/química , Factor Esteroidogénico 1/química , Aminoácidos/química , Animales , Transporte Biológico , Núcleo Celular/metabolismo , Cromatografía , Simulación por Computador , Cristalografía por Rayos X , Electrones , Humanos , Ligandos , Lípidos/química , Ratones , Modelos Moleculares , Conformación Molecular , Mutación , Mutación Missense , Péptidos/química , Transducción de Señal , Solventes/química , Resonancia por Plasmón de Superficie , Propiedades de Superficie , Temperatura , Agua/químicaRESUMEN
The crystal structure of arabinose-5-phosphate isomerase (API) from Bacteroides fragilis (bfAPI) was determined at 1.7â Å resolution and was found to be a tetramer of a single-domain sugar isomerase (SIS) with an endogenous ligand, CMP-Kdo (cytidine 5'-monophosphate-3-deoxy-D-manno-oct-2-ulosonate), bound at the active site. API catalyzes the reversible isomerization of D-ribulose 5-phosphate to D-arabinose 5-phosphate in the first step of the Kdo biosynthetic pathway. Interestingly, the bound CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product in the Kdo biosynthetic pathway and presumably acts as a feedback inhibitor for bfAPI. The active site of each monomer is located in a surface cleft at the tetramer interface between three monomers and consists of His79 and His186 from two different adjacent monomers and a Ser/Thr-rich region, all of which are highly conserved across APIs. Structure and sequence analyses indicate that His79 and His186 may play important catalytic roles in the isomerization reaction. CMP-Kdo mimetics could therefore serve as potent and specific inhibitors of API and provide broad protection against many different bacterial infections.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/metabolismo , Bacteroides fragilis/química , Isomerasas Aldosa-Cetosa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Citidina Monofosfato/análogos & derivados , Citidina Monofosfato/química , Histidina/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Azúcares Ácidos/químicaRESUMEN
Kanadaptin is a nuclear protein of unknown function that is widely expressed in mammalian tissues. The crystal structure of the forkhead-associated (FHA) domain of human kanadaptin was determined to 1.6 Å resolution. The structure reveals an asymmetric dimer in which one monomer is complexed with a phosphopeptide mimic derived from a peptide segment from the N-terminus of a symmetry-related molecule as well as a sulfate bound to the structurally conserved phosphothreonine recognition cleft. This structure provides insights into the molecular recognition features utilized by this family of proteins and represents the first evidence that kanadaptin is likely involved in a phosphorylation-mediated signaling pathway. These results will be of use for designing experiments to further probe the function of kanadaptin.
Asunto(s)
Antiportadores/química , Antiportadores/metabolismo , Fosfopéptidos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfopéptidos/química , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Crystal structures of three members (BACOVA_00364 from Bacteroides ovatus, BACUNI_03039 from Bacteroides uniformis and BACEGG_00036 from Bacteroides eggerthii) of the Pfam domain of unknown function (DUF4488) were determined to 1.95, 1.66, and 1.81 Å resolutions, respectively. The protein structures adopt an eight-stranded, calycin-like, ß-barrel fold and bind an endogenous unknown ligand at one end of the ß-barrel. The amino acids interacting with the ligand are not conserved in any other protein of known structure with this particular fold. The size and chemical environment of the bound ligand suggest binding or transport of a small polar molecule(s) as a potential function for these proteins. These are the first structural representatives of a newly defined PF14869 (DUF4488) Pfam family.
Asunto(s)
Proteínas Bacterianas/química , Bacteroides/metabolismo , Metabolismo de los Hidratos de Carbono , Bacteroides/química , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de SecuenciaRESUMEN
Pyridoxal-5'-phosphate or PLP, the active form of vitamin B6, is a highly versatile cofactor that participates in a large number of mechanistically diverse enzymatic reactions in basic metabolism. PLP-dependent enzymes account for â¼1.5% of most prokaryotic genomes and are estimated to be involved in â¼4% of all catalytic reactions, making this an important class of enzymes. Here, we structurally and functionally characterize three novel PLP-dependent enzymes from bacteria in the human microbiome: two are from Eubacterium rectale, a dominant, nonpathogenic, fecal, Gram-positive bacteria, and the third is from Porphyromonas gingivalis, which plays a major role in human periodontal disease. All adopt the Type I PLP-dependent enzyme fold and structure-guided biochemical analysis enabled functional assignments as tryptophan, aromatic, and probable phosphoserine aminotransferases.
Asunto(s)
Eubacterium/enzimología , Microbiota , Oxidorreductasas/metabolismo , Porphyromonas gingivalis/enzimología , Fosfato de Piridoxal/metabolismo , Transaminasas/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Oxidorreductasas/química , Conformación Proteica , Fosfato de Piridoxal/química , Transaminasas/químicaRESUMEN
PF10014 is a novel family of 2-oxyglutarate-Fe(2+) -dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the ß-strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site.