RESUMO
Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu â Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.
Assuntos
Anemia Falciforme/tratamento farmacológico , Hemoglobina A/efeitos dos fármacos , Hemoglobina Falciforme/efeitos dos fármacos , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Animais , Eritrócitos/metabolismo , Camundongos , Oxigênio/metabolismo , Quinolinas/químicaRESUMO
Increased fructose consumption and its subsequent metabolism have been implicated in metabolic disorders such as nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) and insulin resistance. Ketohexokinase (KHK) converts fructose to fructose-1-phosphate (F1P) in the first step of the metabolic cascade. Herein we report the discovery of a first-in-class KHK inhibitor, PF-06835919 (8), currently in phase 2 clinical trials. The discovery of 8 was built upon our originally reported, fragment-derived lead 1 and the recognition of an alternative, rotated binding mode upon changing the ribose-pocket binding moiety from a pyrrolidinyl to an azetidinyl ring system. This new binding mode enabled efficient exploration of the vector directed at the Arg-108 residue, leading to the identification of highly potent 3-azabicyclo[3.1.0]hexane acetic acid-based KHK inhibitors by combined use of parallel medicinal chemistry and structure-based drug design.
Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Frutoquinases/antagonistas & inibidores , Frutoquinases/metabolismo , Frutose/efeitos adversos , Doenças Metabólicas/enzimologia , Animais , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Frutose/administração & dosagem , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Resistência à Insulina/fisiologia , Masculino , Doenças Metabólicas/induzido quimicamente , Doenças Metabólicas/tratamento farmacológico , Estrutura Secundária de Proteína , Ratos , Ratos WistarRESUMO
Many diseases are believed to be driven by pathological levels of reactive oxygen species (ROS) and oxidative stress has long been recognized as a driver for inflammatory disorders. Apoptosis signal-regulating kinase 1 (ASK1) has been reported to be activated by intracellular ROS and its inhibition leads to a down regulation of p38-and JNK-dependent signaling. Consequently, ASK1 inhibitors may have the potential to treat clinically important inflammatory pathologies including renal, pulmonary and liver diseases. Analysis of the ASK1 ATP-binding site suggested that Gln756, an amino acid that rarely occurs at the GK+2 position, offered opportunities for achieving kinase selectivity for ASK1 which was applied to the design of a parallel medicinal chemistry library that afforded inhibitors of ASK1 with nanomolar potency and excellent kinome selectivity. A focused optimization strategy utilizing structure-based design resulted in the identification of ASK1 inhibitors with low nanomolar potency in a cellular assay, high selectivity when tested against kinase and broad pharmacology screening panels, and attractive physicochemical properties. The compounds we describe are attractive tool compounds to inform the therapeutic potential of ASK1 inhibition.
Assuntos
Amidas/farmacologia , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Amidas/síntese química , Amidas/química , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.
Assuntos
Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteoma/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Sequência de Aminoácidos , Animais , Área Sob a Curva , Encéfalo/metabolismo , Cristalografia por Raios X , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutação de Sentido Incorreto , Nitrilas/química , Nitrilas/farmacocinética , Doença de Parkinson/tratamento farmacológico , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteoma/química , Proteoma/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirróis/química , Pirróis/farmacocinética , RatosRESUMO
Acid-sensing ion channels (ASICs) are voltage-independent, proton-activated receptors that belong to the epithelial sodium channel/degenerin family of ion channels and are implicated in perception of pain, ischaemic stroke, mechanosensation, learning and memory. Here we report the low-pH crystal structure of a chicken ASIC1 deletion mutant at 1.9 A resolution. Each subunit of the chalice-shaped homotrimer is composed of short amino and carboxy termini, two transmembrane helices, a bound chloride ion and a disulphide-rich, multidomain extracellular region enriched in acidic residues and carboxyl-carboxylate pairs within 3 A, suggesting that at least one carboxyl group bears a proton. Electrophysiological studies on aspartate-to-asparagine mutants confirm that these carboxyl-carboxylate pairs participate in proton sensing. Between the acidic residues and the transmembrane pore lies a disulphide-rich 'thumb' domain poised to couple the binding of protons to the opening of the ion channel, thus demonstrating that proton activation involves long-range conformational changes.
Assuntos
Galinhas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido , Animais , Sítios de Ligação , Linhagem Celular , Galinhas/genética , Cloretos/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Proteínas de Membrana/genética , Modelos Moleculares , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Prótons , Deleção de Sequência , Canais de Sódio/genética , Relação Estrutura-AtividadeRESUMO
The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.
Assuntos
Fosfolipases A/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Cisteína , DNA Complementar/genética , Dimerização , Dissulfetos/análise , Venenos Elapídicos/química , Elapidae , Luz , Substâncias Macromoleculares/química , Modelos Moleculares , Fosfolipases A/genética , Fosfolipases A/metabolismo , Fosfolipases A2 , Conformação Proteica , Estrutura Secundária de Proteína , Espalhamento de RadiaçãoRESUMO
This is the first evidence of a naturally bound fatty acid to a group I Phospholipase A(2) (PLA(2)) and also to a PLA(2) with Asp 49. The fatty acid identified as n-tridecanoic acid is observed at the substrate recognition site of PLA(2) hydrophobic channel. The complex was isolated from the venom of Bungarus caeruleus (Common Indian Krait). The primary sequence of the PLA(2) was determined using the cDNA method. Three-dimensional structure has been solved by the molecular replacement method and refined using the CNS package to a final R factor of 19.8% for the data in the resolution range from 20.0 to 2.7 A. The final refined model is comprised of 912 protein atoms, one sodium ion, one molecule of n-tridecanoic acid, and 60 water molecules. The sodium ion is located in the calcium-binding loop with a sevenfold coordination. A characteristic extra electron density was observed in the hydrophobic channel of the enzyme, into which a molecule of n-tridecanoic acid was clearly fitted. The MALDI-TOF measurements of the crystals had earlier indicated an increase in the molecular mass of PLA(2) by 212 Da over the native PLA(2). A major part of the ligand fits well in the binding pocket and interacts directly with His 48 and Asp 49. Although the overall structure of PLA(2) in the present complex is similar to the native structure reported earlier, it differs significantly in the folding of its calcium-binding loop.
Assuntos
Ácidos Graxos/química , Fosfolipases A/química , Alcanos/química , Sequência de Aminoácidos , Animais , Ácido Aspártico/química , Bungarus/metabolismo , Cálcio/química , Cristalografia por Raios X , DNA Complementar/metabolismo , Ácidos Dicarboxílicos/química , Elétrons , Histidina/química , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipases A2 , Dobramento de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Venenos de Serpentes , Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Echicetin is a heterodimeric protein from the venom of the Indian saw-scaled viper, Echis carinatus. It binds to platelet glycoprotein Ib (GPIb) and thus inhibits platelet aggregation. It has two subunits, alpha and beta, consisting of 131 and 123 amino acid residues, respectively. The two chains are linked with a disulphide bond. The level of amino acid sequence homology between two subunits is 50%. The protein was purified from the venom of E.carinatus and crystallized using ammonium sulphate as a precipitant. The crystal structure has been determined at 2.4A resolution and refined to an R-factor of 0.187. Overall dimensions of the heterodimer are approximately 80Ax35Ax35A. The backbone folds of the two subunits are similar. The central portions of the polypeptide chains of alpha and beta-subunits move into each other to form a tight dimeric association. The remaining portions of the chains of both subunits fold in a manner similar to those observed in the carbohydrate-binding domains of C-type lectins. In echicetin, the Ca(2+)-binding sites are not present, despite being topologically equivalent to other similar Ca(2+)-binding proteins of the superfamily. The residues Ser41, Glu43 and Glu47 in the calcium-binding proteins of the related family are conserved but the residues Glu126/120 are replaced by lysine at the corresponding sites in the alpha and beta-subunits.
Assuntos
Proteínas/química , Venenos de Víboras/química , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte , Sequência Conservada , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Subunidades Proteicas/química , Proteínas/isolamento & purificaçãoRESUMO
The crystal structure of an acidic phospholipase A(2) from the venom of Echis carinatus (saw-scaled viper; scPLA(2)) has been determined at 2.6 A resolution and refined to a crystallographic R factor of 0.192. Although the overall structure of scPLA(2) is essentially similar to those of other group II acidic PLA(2)s from different species, it shows unique features in several parts. Particularly noteworthy is the C-terminal part, which folds differently to those of other group II PLA(2)s. This part is considered to be responsible for inhibition of the platelet-aggregation activity. The calcium-binding loop is tightly organized with sevenfold coordination. Another striking feature of scPLA(2) is the involvement of Asn79 O(delta1) of a symmetry-related molecule in a coordination linkage with Ca(2+) of the calcium-binding loop. This is the first observation of an internal metal ion participating in an intermolecular interaction. The beta-wing of a molecule is deeply inserted into the hydrophobic channel of another molecule and forms several intermolecular interactions. This results in the formation of an infinite chain of molecules. These chains are stacked in an antiparallel arrangement in the crystals.
Assuntos
Fosfolipases A/química , Viperidae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipases A/genética , Fosfolipases A/farmacologia , Fosfolipases A2 , Alinhamento de Sequência , Análise de Sequência de DNA , Propriedades de SuperfícieRESUMO
This is the first crystal structure of a proteolytically generated functional C-lobe of lactoferrin. The purified samples of iron-saturated C-lobe were crystallized in 0.1 M Mes buffer (pH 6.5) containing 25% (v/v) polyethyleneglycol monomethyl ether 550 M and 0.1 M zinc sulphate heptahydrate. The X-ray intensity data were collected with 300 mm imaging plate scanner mounted on a rotating anode generator. The structure was determined by the molecular replacement method using the coordinates of the C-terminal half of bovine lactoferrin as a search model and refined to an R-factor of 0.193 for all data to 1.9A resolution. The final model comprises 2593 protein atoms (residues 342-676 and 681-685), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), one Fe(3+), one CO(3)(2-), two Zn(2+) and 230 water molecules. The overall folding of the C-lobe is essentially the same as that of C-terminal half of bovine lactoferrin but differs slightly in conformations of some of the loops and reveals a number of new interactions. There are 20 Cys residues in the C-lobe forming ten disulphide links. Out of these, one involving Cys481-Cys675 provides an inter-domain link at 2.01A while another Cys405-Cys684 is formed between the main C-lobe 342-676 and the hydrolyzed pentapeptide 681-685 fragment. Six inter-domain hydrogen bonds have been observed in the structure whereas only four were reported in the structure of intact lactoferrin, although domain orientations have been found similar in the two structures. The good quality of electron density has also revealed all the ten oligosaccharide units in the structure. The observation of two metal ions at sites other than the iron-binding cleft is another novel feature of the present structure. These zinc ions stabilize the crystal packing. This structure is also notable for extensive inter-molecular hydrogen bonding in the crystals. Therefore, the present structure appears to be one of the best packed crystal structures among the proteins of the transferrin superfamily.
Assuntos
Lactoferrina/química , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Cisteína/química , Cistina/química , Elétrons , Hidrogênio/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Íons , Ferro , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
Phospholipase A(2) (EC 3.1.1.4) is a key enzyme of the cascade mechanism involved in the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to membrane surfaces and the hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. In order to regulate the production of proinflammatory compounds, a specific peptide inhibitor of PLA(2), Leu-Ala-Ile-Tyr-Ser, has been designed. Phospholipase A(2) from Daboia russelli pulchella (DPLA(2)) and peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) have been co-crystallized. The structure of the complex has been determined and refined to 2.0 A resolution. The structure contains two crystallographically independent molecules of DPLA(2), with one molecule of peptide specifically bound to one of them. The overall conformations of the two molecules are essentially similar except in three regions; namely, the calcium-binding loop including Trp31 (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Of these, the most striking difference pertains to the orientation of Trp31 in the two molecules. The conformation of Trp31 in molecule A was suitable to allow the binding of peptide LAIYS, while that in molecule B prevented the entry of the ligand into the hydrophobic channel. The structure of the complex clearly showed that the OH group of Tyr of the inhibitor formed hydrogen bonds with both His48 N(delta1) and Asp49 O(delta1), while O(gamma)H of Ser was involved in a hydrogen bond with Trp31. Other peptide backbone atoms interact with protein through water molecules, while Leu, Ala and Ile form strong hydrophobic interactions with the residues of the hydrophobic channel.
Assuntos
Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fosfolipases A/antagonistas & inibidores , Venenos de Víboras/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Luz , Modelos Moleculares , Fosfolipases A/química , Fosfolipases A2 , Conformação Proteica , Estrutura Secundária de Proteína , Daboia , Espalhamento de Radiação , TriptofanoRESUMO
This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).
Assuntos
Aristolochia/química , Ácidos Aristolóquicos , Inibidores Enzimáticos/química , Fenantrenos/química , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/química , Prostaglandinas/biossíntese , Venenos de Víboras/antagonistas & inibidores , Venenos de Víboras/enzimologia , Animais , Ligação Competitiva , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Fosfolipases A2 , Conformação Proteica , Daboia , Relação Estrutura-AtividadeRESUMO
Phospholipase A(2) is an important enzyme involved in the production of prostaglandins and their related compounds causing inflammatory disorders. Among the several peptides tested, the peptide Phe-Leu-Ser-Tyr-Lys (FLSYK) showed the highest inhibition. The dissociation constant (K(d)) for this peptide was calculated to be 3.57 +/- 0.05 x 10(-9) m. In order to further improve the degree of inhibition of phospholipase A(2), a complex between Russells viper snake venom phospholipase A(2) and a peptide inhibitor FLSYK was crystallized, and its structure was determined by crystallographic methods and refined to an R-factor of 0.205 at 1.8 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) (molecules A and B) and a peptide molecule specifically bound to molecule A only. The two molecules formed an asymmetric dimer. The dimerization caused a modification in the binding site of molecule A. The overall conformations of molecules A and B were found to be generally similar except three regions i.e. the Trp-31-containing loop (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Out of the above three, the most striking difference pertains to the conformation of Trp-31 in the two molecules. The orientation of Trp-31 in molecule A was suitable for the binding of FLSYK, while it disallowed the binding of peptide to molecule B. The structure of the complex clearly shows that the peptide is so placed in the binding site of molecule A that the side chain of its lysine residue interacted extensively with the enzyme and formed several hydrogen bonds in addition to a strong electrostatic interaction with critical Asp-49. The C-terminal carboxylic group of the peptide interacted with the catalytic residue His-48.
Assuntos
Inibidores Enzimáticos/química , Oligopeptídeos/química , Fosfolipases A/química , Venenos de Víboras/enzimologia , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/farmacologia , Fosfolipases A/antagonistas & inibidoresRESUMO
This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex.