RESUMO
The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.
Assuntos
Trifosfato de Adenosina/análogos & derivados , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Dependovirus/genética , Proteínas Virais/genética , Trifosfato de Adenosina/genética , Microscopia Crioeletrônica , DNA Helicases/genética , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Dependovirus/ultraestrutura , Humanos , Proteínas Virais/ultraestruturaRESUMO
Sickle cell disease (SCD) is caused by a single-point mutation, and the ensuing deoxygenation-induced polymerization of sickle hemoglobin (HbS), and reduction in bioavailability of vascular nitric oxide (NO), contribute to the pathogenesis of the disease. In a proof-of-concept study, we successfully incorporated nitrate ester groups onto two previously studied potent antisickling aromatic aldehydes, TD7 and VZHE039, to form TD7-NO and VZHE039-NO hybrids, respectively. These compounds are stable in buffer but demonstrated the expected release of NO in whole blood in vitro and in mice. The more promising VZHE039-NO retained the functional and antisickling activities of the parent VZHE039 molecule. Moreover, VZHE039-NO, unlike VZHE039, significantly attenuated RBC adhesion to laminin, suggesting this compound has potential in vivo RBC anti-adhesion properties relevant to vaso-occlusive events. Crystallographic studies show that, as with VZHE039, VZHE039-NO also binds to liganded Hb to make similar protein interactions. The knowledge gained during these investigations provides a unique opportunity to generate a superior candidate drug in SCD with enhanced benefits.
Assuntos
Anemia Falciforme , Hemoglobina Falciforme , Camundongos , Animais , Hemoglobina Falciforme/metabolismo , Antidrepanocíticos/farmacologia , Antidrepanocíticos/uso terapêutico , Óxido Nítrico , Aldeídos/farmacologia , Nitratos , Laminina , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/metabolismo , ÉsteresRESUMO
Two natural products, compounds 1 and 2 were isolated from the root bark of Ziziphus abyssinica for the first time and were structurally elucidated as ß-amyrin and polpunonic acid, respectively. Both compounds were further subjected to an in vivo study in rats to evaluate their anti-arthritic potency. Compared to the arthritic control group, rats treated with different doses of 1 or 2 (3, 10, and 30 mg/kg) exhibited significantly higher total change in body weight as well as lower arthritic scores and total change in paw edema and erythema. Histopathological examinations of the hind paws of the rats further demonstrated the beneficial effects of both compounds as they significantly reversed cartilage erosion, subchondral cyst, and Weichselbaum's lacunae formation. Evidence of bone remodeling was also observed in all groups of rats treated with 1 or 2. Hematological and serum biochemical parameters were not significantly affected by treatment of 1 or 2. Taken together, the results from the present study suggest potential therapeutic benefit of ß-amyrin and polpunonic acid in rheumatoid arthritis and related inflammatory disorders.
Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Ácido Oleanólico/análogos & derivados , Rhamnaceae/química , Triterpenos/farmacologia , Analgésicos/química , Analgésicos/isolamento & purificação , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Artrite Experimental/induzido quimicamente , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Edema/tratamento farmacológico , Feminino , Adjuvante de Freund/administração & dosagem , Masculino , Estrutura Molecular , Ácido Oleanólico/química , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologia , Casca de Planta/química , Raízes de Plantas/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Triterpenos/química , Triterpenos/isolamento & purificaçãoRESUMO
Certain 2-amino-3,4-dihydroquinazolines bind at 5-HT3 serotonin receptors and act as antagonists (e.g. 6-chloro) whereas others bind with little to no affinity and lack functional activity (e.g. 8-chloro). The purpose of this investigation was to gain insight as to why this might be the case. X-Ray crystallographic studies revealed that the N-C-N distances in the examined analogs are nearly identical (1.31 - 1.34 Å), suggesting that differences in N-C-N delocalization does not account for differences in affinity/action. Homology modeling hydrophatic interactions (HINT) analysis revealed that the 6-chloro analog formed a greater number, and more favorable, interactions with the receptor, whereas the 8-chloro analog formed fewer, and unfavorable, interactions. The affinity and activity of the 6-chloro quinazoline relative to its 8-chloro counterpart are unrelated to the N-C-N delocalization pattern but might be related to specific (favorable and unfavorable) interactions of quinazoline substituents with certain receptor features as determined by HINT analysis.
RESUMO
Sickle cell disease is an inherited disorder of hemoglobin (Hb). During a sickle cell crisis, deoxygenated sickle hemoglobin (deoxyHbS) polymerizes to form fibers in red blood cells (RBCs), causing the cells to adopt "sickled" shapes. Using small molecules to increase the affinity of Hb for oxygen is a potential approach to treating sickle cell disease, because oxygenated Hb interferes with the polymerization of deoxyHbS. We have identified a triazole disulfide compound (4,4'-di(1,2,3-triazolyl)disulfide, designated TD-3), which increases the affinity of Hb for oxygen. The crystal structures of carboxy- and deoxy-forms of human adult Hb (HbA), each complexed with TD-3, revealed that one molecule of the monomeric thiol form of TD-3 (5-mercapto-1H-1,2,3-triazole, designated MT-3) forms a disulfide bond with ß-Cys93, which inhibits the salt-bridge formation between ß-Asp94 and ß-His146. This inhibition of salt bridge formation stabilizes the R-state and destabilizes the T-state of Hb, resulting in reduced magnitude of the Bohr effect and increased affinity of Hb for oxygen. Intravenous administration of TD-3 (100 mg/kg) to C57BL/6 mice increased the affinity of murine Hb for oxygen, and the mice did not appear to be adversely affected by the drug. TD-3 reduced in vitro hypoxia-induced sickling of human sickle RBCs. The percentage of sickled RBCs and the P50 of human SS RBCs by TD-3 were inversely correlated with the fraction of Hb modified by TD-3. Our study shows that TD-3, and possibly other triazole disulfide compounds that bind to Hb ß-Cys93, may provide new treatment options for patients with sickle cell disease.
Assuntos
Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/farmacologia , Dissulfetos/farmacologia , Eritrócitos/efeitos dos fármacos , Hemoglobinas/metabolismo , Oxigênio/metabolismo , Triazóis/farmacologia , Anemia Falciforme/metabolismo , Animais , Eritrócitos/metabolismo , Hemoglobina Falciforme/metabolismo , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/metabolismo , Metalotioneína 3 , Camundongos , Camundongos Endogâmicos C57BL , Polimerização/efeitos dos fármacos , Ligação ProteicaRESUMO
Hypoxia-induced polymerization of sickle hemoglobin (Hb S) is the principal phenomenon that underlays the pathophysiology and morbidity associated with sickle cell disease (SCD). Opportunely, as an allosteric protein, hemoglobin (Hb) serves as a convenient and potentially critical druggable target. Consequently, molecules that prevent Hb S polymerization (Hb modifiers), and the associated erythrocyte sickling have been investigated-and retain significant interest-as a viable therapeutic strategy for SCD. This group of molecules, including aromatic aldehydes, form high oxygen affinity Schiff-base adducts with Hb S, which are resistant to polymerization. Here, we report the design and synthesis of novel potent antisickling agents (SAJ-009, SAJ-310 and SAJ-270) based on the pharmacophore of vanillin and INN-312, a previously reported pyridyl derivative of vanillin. These novel derivatives exhibited superior in vitro binding and pharmacokinetic properties compared to vanillin, which translated into significantly enhanced allosteric and antisickling properties. Crystal structure studies of liganded Hb in the R2 quaternary state in complex with SAJ-310 provided important insights into the allosteric and antisickling properties of this group of compounds. While these derivatives generally show similar in vitro biological potency, significant structure-dependent differences in their biochemical profiles would help predict the most promising candidates for successful in vivo pre-clinical translational studies and inform further structural modifications to improve on their pharmacologic properties.
Assuntos
Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/farmacologia , Benzaldeídos/farmacologia , Hemoglobina Falciforme/metabolismo , Piridinas/farmacologia , Antidrepanocíticos/síntese química , Antidrepanocíticos/química , Antidrepanocíticos/metabolismo , Benzaldeídos/síntese química , Benzaldeídos/química , Benzaldeídos/metabolismo , Sangue/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Piridinas/síntese química , Piridinas/química , Piridinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Candidate drugs to counter intracellular polymerization of deoxygenated sickle hemoglobin (Hb S) continue to represent a promising approach to mitigating the primary cause of the pathophysiology associated with sickle cell disease (SCD). One such compound is the naturally occurring antisickling agent, 5-hydroxymethyl-2-furfural (5-HMF), which has been studied in the clinic for the treatment of SCD. As part of our efforts to develop novel efficacious drugs with improved pharmacologic properties, we structurally modified 5-HMF into 12 ether and ester derivatives. The choice of 5-HMF as a pharmacophore was influenced by a combination of its demonstrated attractive hemoglobin modifying and antisickling properties, well-known safety profiles, and its reported nontoxic major metabolites. The derivatives were investigated for their time- and/or dose-dependent effects on important antisickling parameters, such as modification of hemoglobin, corresponding changes in oxygen affinity, and inhibition of red blood cell sickling. The novel test compounds bound and modified Hb and concomitantly increased the protein affinity for oxygen. Five of the derivatives exhibited 1.5- to 4.0-fold higher antisickling effects than 5-HMF. The binding mode of the compounds with Hb was confirmed by X-ray crystallography and, in part, helps explain their observed biochemical properties. Our findings, in addition to the potential therapeutic application, provide valuable insights and potential guidance for further modifications of these (and similar) compounds to enhance their pharmacologic properties.
Assuntos
Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/farmacologia , Desenho de Fármacos , Furaldeído/análogos & derivados , Hemoglobina Falciforme/metabolismo , Anemia Falciforme/sangue , Antidrepanocíticos/síntese química , Antidrepanocíticos/uso terapêutico , Química Farmacêutica , Cristalização , Cristalografia por Raios X , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Ésteres/química , Éteres/química , Furaldeído/química , Furaldeído/farmacologia , Furaldeído/uso terapêutico , Voluntários Saudáveis , Humanos , Modelos Moleculares , Oxigênio/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Fatores de Tempo , Resultado do TratamentoRESUMO
Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains.
Assuntos
Proteínas de Ligação a DNA/química , Dependovirus/fisiologia , Proteínas Virais/química , Integração Viral , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , DNA Viral/química , Proteínas de Ligação a DNA/metabolismo , Dependovirus/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Virais/metabolismoRESUMO
L-Threonine aldolases (TAs), a family of enzymes belonging to the fold-type I pyridoxal 5'-phosphate (PLP) dependent enzymes, play a role in catalyzing the reversible cleavage of l-3-hydroxy-α-amino acids to glycine and the corresponding aldehydes. Threonine aldolases have great biotechnological potential for the syntheses of pharmaceutically relevant drug molecules because of their stereospecificity. The pH-dependency of their catalytic activity, affecting reaction intermediates, led us to study the effect of low-pH on Escherichia coli TA (eTA) structure. We report here a low-pH crystal structure of eTA at 2.1 Å resolution, with a non-covalently bound uncleaved l-serine substrate, and a PLP cofactor bound as an internal aldimine. This structure contrasts with other eTA structures obtained at physiological pH that show products or substrates bound as PLP-external aldimines. The non-productive binding at low-pH is due to an unusual substrate serine binding orientation in which the α-amino group and carboxylate group are in the wrong positions (relative to the active site residues) as a result of protonation of the α-amino group of the serine, as well as the active site histidines, His83 and His126. Protonation of these residues prevents the characteristic nucleophilic attack of the α-amino group of substrate serine on C4' of PLP to form the external aldimine. Our study shows that at low pH the change in charge distribution at the active site can result in substrates binding in a non-productive orientation.
Assuntos
Escherichia coli/enzimologia , Glicina Hidroximetiltransferase/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Glicina Hidroximetiltransferase/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ligação Proteica , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismoRESUMO
Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of â¼20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.
Assuntos
Proteínas de Ligação a DNA/química , Dependovirus/química , Proteínas Virais/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Infecções por Parvoviridae/virologia , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to ß-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression. We solved the crystal structure of OrfX at 1.7 Å and found that it belongs to the S-adenosyl-L-methionine (AdoMet)-dependent α/ß-knot superfamily of SPOUT methyltransferases (MTases), with a high structural homology to YbeA, the gene product of the Escherichia coli 70 S ribosomal MTase RlmH. MTase activity was confirmed by demonstrating the OrfX-dependent methylation of the Staphylococcus aureus 70 S ribosome. When OrfX was crystallized in the presence of its AdoMet substrate, we found that each monomer of the homodimeric structure bound AdoMet in its active site. Solution studies using isothermal titration calorimetry confirmed that each monomer bound AdoMet but with different binding affinities (K(d) = 52 ± 0.4 and 606 ± 2 µm). In addition, the structure shows that the AdoMet-binding pocket, formed by a deep trefoil knot, contains a bound phosphate molecule, which is the likely nucleotide methylation site. This study represents the first characterization of a staphylococcal ribosomal MTase and provides the first crystal structure of a member of the α/ß-knot superfamily of SPOUT MTases in the RlmH or COG1576 family with bound AdoMet.
Assuntos
Proteínas de Bactérias/metabolismo , Metiltransferases/química , RNA Ribossômico/metabolismo , Staphylococcus aureus/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Cinética , Metiltransferases/genética , Modelos Genéticos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por SubstratoRESUMO
Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function.
Assuntos
Piridoxina , Vitamina B 6 , Vitamina B 6/química , Piridoxina/metabolismo , Escherichia coli/metabolismo , Fosfato de Piridoxal/metabolismo , Piridoxal/metabolismoRESUMO
The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.
Assuntos
Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/química , Metiltransferases/química , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/genética , Cristalografia por Raios X , Metilação de DNA , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Humanos , Metiltransferases/deficiência , Metiltransferases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Subunidades Ribossômicas Menores de Bactérias/enzimologia , Subunidades Ribossômicas Menores de Bactérias/genética , Especificidade por Substrato/genéticaRESUMO
Hsp70s are ubiquitous and highly conserved molecular chaperones. They play crucial roles in maintaining cellular protein homeostasis. It is well established that Hsp70s use the energy of ATP hydrolysis to ADP to power the chaperone activity regardless of the cellular locations and isoforms. Binding immunoglobin protein (BiP), the major member of Hsp70s in the endoplasmic reticulum, is essential for protein folding and quality control. Unexpectedly, our structural analysis of BiP demonstrated a novel ATP hydrolysis to AMP during crystallization under the acidic conditions. Our biochemical studies confirmed this newly discovered ATP to AMP hydrolysis in solutions. Unlike the canonical ATP to ADP hydrolysis observed for Hsp70s, this ATP hydrolysis to AMP depends on the substrate-binding domain of BiP and is inhibited by the binding of a peptide substrate. Intriguingly, this ATP to AMP hydrolysis is unique to BiP, not shared by two representative Hsp70 proteins from the cytosol. Taken together, this novel and unique ATP to AMP hydrolysis may provide a potentially new direction for understanding the activity and cellular function of BiP.
Assuntos
Proteínas de Transporte , Proteínas de Choque Térmico HSP70 , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70/química , Humanos , Hidrólise , Ligação ProteicaRESUMO
Bisphosphoglycerate mutase (BPGM) is an erythrocyte-specific multifunctional enzyme that is responsible for the regulation of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells through its synthase and phosphatase activities; the latter enzymatic function is stimulated by the endogenous activator 2-phosphoglycolate (2-PG). 2,3-BPG is a natural allosteric effector of hemoglobin (Hb) that is responsible for decreasing the affinity of Hb for oxygen to facilitate tissue oxygenation. Here, crystal structures of BPGM with 2-PG in the presence and absence of 3-phosphoglycerate are reported at 2.25 and 2.48â Å resolution, respectively. Structure analysis revealed a new binding site for 2-PG at the dimer interface for the first time, in addition to the expected active-site binding. Also, conformational non-equivalence of the two active sites was observed as one of the sites was found in an open conformation, with the residues at the active-site entrance, including Arg100, Arg116 and Arg117, and the C-terminus disordered. The kinetic result is consistent with the binding of 2-PG to an allosteric or noncatalytic site as well as the active site. This study paves the way for the rational targeting of BPGM for therapeutic purposes, especially for the treatment of sickle cell disease.
Assuntos
Bisfosfoglicerato Mutase , Glicolatos , Sítios de Ligação , Glicolatos/metabolismo , Monoéster Fosfórico HidrolasesRESUMO
5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies.
Assuntos
Anemia Falciforme , Pró-Fármacos , Hemoglobinas/química , Humanos , Óxido Nítrico , Oxigênio/química , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêuticoRESUMO
The pyridoxal 5'-phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP-dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP-dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Lisina/metabolismo , Fosfato de Piridoxal , Proteínas/química , Estabilidade Proteica , Homeostase , Fosfatos/metabolismo , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Heme is an important cofactor in a large number of essential proteins and is often involved in small molecule binding and activation. Loss of heme from proteins thus negatively affects the function of these proteins but is also an important component of iron recycling. The characterization of intermediates that form during the loss of heme from proteins has been problematic, in a large part, because of the instability of such intermediates. We have characterized, by X-ray crystallography, three compounds that form during the nitrite-induced degradation of human α(2)ß(2) hemoglobin (Hb). The first is an unprecedented complex that exhibits a large ß heme displacement of 4.8 Å toward the protein exterior; the heme displacement is stabilized by the binding of the distal His residue to the heme Fe, which in turn allows for the unusual binding of an exogenous ligand on the proximal face of the heme. We have also structurally characterized complexes that display regiospecific nitration of the heme at the 2-vinyl position; we show that heme nitration is not a prerequisite for heme loss. Our results provide structural insight into a possible pathway for nitrite-induced loss of heme from human Hb.
Assuntos
Heme/química , Hemoglobinas/química , Nitritos/farmacologia , Cristalografia por Raios X , Heme/metabolismo , Hemoglobinas/metabolismo , Histidina/química , Humanos , Modelos Moleculares , Nitritos/químicaRESUMO
Vanillin has previously been studied clinically as an antisickling agent to treat sickle-cell disease. In vitro investigations with pyridyl derivatives of vanillin, including INN-312 and INN-298, showed as much as a 90-fold increase in antisickling activity compared with vanillin. The compounds preferentially bind to and modify sickle hemoglobin (Hb S) to increase the affinity of Hb for oxygen. INN-312 also led to a considerable increase in the solubility of deoxygenated Hb S under completely deoxygenated conditions. Crystallographic studies of normal human Hb with INN-312 and INN-298 showed that the compounds form Schiff-base adducts with the N-terminus of the α-subunits to constrain the liganded (or relaxed-state) Hb conformation relative to the unliganded (or tense-state) Hb conformation. Interestingly, while INN-298 binds and directs its meta-positioned pyridine-methoxy moiety (relative to the aldehyde moiety) further down the central water cavity of the protein, that of INN-312, which is ortho to the aldehyde, extends towards the surface of the protein. These studies suggest that these compounds may act to prevent sickling of SS cells by increasing the fraction of the soluble high-affinity Hb S and/or by stereospecific inhibition of deoxygenated Hb S polymerization.
Assuntos
Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/química , Benzaldeídos/química , Hemoglobina Falciforme/química , Piridinas/química , Anemia Falciforme/metabolismo , Antidrepanocíticos/farmacologia , Benzaldeídos/metabolismo , Benzaldeídos/uso terapêutico , Cristalização , Cristalografia por Raios X , Hemoglobina Falciforme/metabolismo , Humanos , Oxigênio/química , Oxigênio/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Piridinas/metabolismo , Piridinas/uso terapêutico , Bases de Schiff/química , Solubilidade/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Sickle cell disease (SCD) is associated with hemolysis, vascular inflammation, and organ damage. Affected patients experience chronic painful vaso-occlusive events requiring hospitalization. Hypoxia-induced polymerization of sickle hemoglobin S (HbS) contributes to sickling of red blood cells (RBCs) and disease pathophysiology. Dilution of HbS with nonsickling hemoglobin or hemoglobin with increased oxygen affinity, such as fetal hemoglobin or HbS bound to aromatic aldehydes, is clinically beneficial in decreasing polymerization. We investigated a novel alternate approach to modify HbS and decrease polymerization by inhibiting methionine aminopeptidase 2 (MetAP2), which cleaves the initiator methionine (iMet) from Val1 of α-globin and ßS-globin. Kinetic studies with MetAP2 show that ßS-globin is a fivefold better substrate than α-globin. Knockdown of MetAP2 in human umbilical cord blood-derived erythroid progenitor 2 cells shows more extensive modification of α-globin than ß-globin, consistent with kinetic data. Treatment of human erythroid cells in vitro or Townes SCD mice in vivo with selective MetAP2 inhibitors extensively modifies both globins with N-terminal iMet and acetylated iMet. HbS modification by MetAP2 inhibition increases oxygen affinity, as measured by decreased oxygen tension at which hemoglobin is 50% saturated. Acetyl-iMet modification on ßS-globin delays HbS polymerization under hypoxia. MetAP2 inhibitor-treated Townes mice reach 50% total HbS modification, significantly increasing the affinity of RBCs for oxygen, increasing whole blood single-cell RBC oxygen saturation, and decreasing fractional flow velocity losses in blood rheology under decreased oxygen pressures. Crystal structures of modified HbS variants show stabilization of the nonpolymerizing high O2-affinity R2 state, explaining modified HbS antisickling activity. Further study of MetAP2 inhibition as a potential therapeutic target for SCD is warranted.