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1.
Elife ; 102021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33752801

RESUMEN

Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore.


Asunto(s)
Proteínas Algáceas/genética , Channelrhodopsins/genética , Chlamydomonas reinhardtii/genética , Proteínas Algáceas/química , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cristalografía , Isomerismo , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia
2.
Nat Commun ; 11(1): 6442, 2020 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-33353947

RESUMEN

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone's phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.


Asunto(s)
Antipsicóticos/química , Receptores de Dopamina D2/química , Espiperona/química , Animales , Sitios de Unión , Células HEK293 , Humanos , Ligandos , Ratones , Modelos Moleculares , Unión Proteica
3.
J Appl Crystallogr ; 52(Pt 6): 1280-1288, 2019 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-31798359

RESUMEN

A sample-injection device has been developed at SPring-8 Angstrom Compact Free-Electron Laser (SACLA) for serial femtosecond crystallography (SFX) at atmospheric pressure. Microcrystals embedded in a highly viscous carrier are stably delivered from a capillary nozzle with the aid of a coaxial gas flow and a suction device. The cartridge-type sample reservoir is easily replaceable and facilitates sample reloading or exchange. The reservoir is positioned in a cooling jacket with a temperature-regulated water flow, which is useful to prevent drastic changes in the sample temperature during data collection. This work demonstrates that the injector successfully worked in SFX of the human A2A adenosine receptor complexed with an antagonist, ZM241385, in lipidic cubic phase and for hen egg-white lysozyme microcrystals in a grease carrier. The injection device has also been applied to many kinds of proteins, not only for static structural analyses but also for dynamics studies using pump-probe techniques.

4.
Structure ; 26(1): 7-19.e5, 2018 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-29225076

RESUMEN

Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.


Asunto(s)
Aminopiridinas/química , Azepinas/química , Antagonistas de los Receptores de Orexina/química , Receptores de Orexina/química , Orexinas/química , Sulfonamidas/química , Triazoles/química , Aminopiridinas/metabolismo , Animales , Azepinas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía/métodos , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Enlace de Hidrógeno , Cinética , Simulación de Dinámica Molecular , Antagonistas de los Receptores de Orexina/metabolismo , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Sulfonamidas/metabolismo , Sincrotrones , Termodinámica , Triazoles/metabolismo
5.
Science ; 354(6319): 1552-1557, 2016 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-28008064

RESUMEN

Bacteriorhodopsin (bR) is a light-driven proton pump and a model membrane transport protein. We used time-resolved serial femtosecond crystallography at an x-ray free electron laser to visualize conformational changes in bR from nanoseconds to milliseconds following photoactivation. An initially twisted retinal chromophore displaces a conserved tryptophan residue of transmembrane helix F on the cytoplasmic side of the protein while dislodging a key water molecule on the extracellular side. The resulting cascade of structural changes throughout the protein shows how motions are choreographed as bR transports protons uphill against a transmembrane concentration gradient.


Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/ultraestructura , Imagenología Tridimensional , Cristalografía , Citoplasma/química , Rayos Láser , Películas Cinematográficas , Conformación Proteica en Hélice alfa , Protones , Retinaldehído/química , Análisis Espectral
6.
J Biosci Bioeng ; 122(3): 270-5, 2016 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-27215832

RESUMEN

Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins, producing the corresponding epoxides. The H-Lyases have been classified into A, B and C subtypes based on amino acid sequence similarities. These enzymes have attracted much attention as industrial catalysts in the synthesis of chiral chemicals from prochiral halohydrins. In the present study, we constructed mutants of B-type H-Lyase from Corynebacterium sp. N-1074 (HheB) displaying higher enantioselectivity by structure-based site-directed mutagenesis and random mutagenesis. A triple mutant of HheB exhibited 98.5% enantioselectivity, the highest ever reported, toward (R)-4-chloro-3-hydroxy-butyronitrile production, with the yield reaching approximately two-fold that of the wild-type enzyme. We discuss the structural basis of the high enantioselectivity and productivity of the mutant by comparing the crystal structures of the mutant HheB and the wild-type enzyme in complex with or without the substrate analogue.


Asunto(s)
Corynebacterium/enzimología , Liasas/genética , Liasas/metabolismo , Mutagénesis , Proteínas Mutantes/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Corynebacterium/genética , Cristalografía por Rayos X , Compuestos Epoxi/metabolismo , Liasas/química , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Especificidad por Sustrato
7.
Extremophiles ; 20(4): 385-93, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27094682

RESUMEN

Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus.


Asunto(s)
Isomerasas de Aminoácido/química , Proteínas Arqueales/química , Thermoplasmales/enzimología , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Estabilidad de Enzimas , Especificidad por Sustrato , Thermoplasmales/genética
8.
Chem Sci ; 6(11): 6280-6294, 2015 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-26508996

RESUMEN

Nitrile hydratases (NHases) are mononuclear nonheme enzymes that catalyze the hydration of nitriles to amides. NHase is unusual in that it utilizes a low-spin (LS) FeIII center and a unique ligand set comprised of two deprotonated backbone amides, cysteine-based sulfenic acid (RSO(H)) and sulfinic acid (RSO2-), and an unmodified cysteine trans to an exogenous ligand site. Electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD) and low-temperature absorption (LT-Abs) spectroscopies are used to determine the geometric and electronic structures of butyrate-bound (NHaseBA) and active (NHaseAq) NHase. These data calibrate DFT models, which are then extended to explore the mechanism of nitrile hydration by NHase. In particular, the nitrile is activated by coordination to the LS FeIII and the sulfenate group is found to be deprotonated and a significantly better nucleophile than water that can attack the coordinated nitrile to form a cyclic species. Attack at the sulfenate S atom of the cyclic species is favorable and leads to a lower kinetic barrier than attack by water on coordinated, uncyclized nitrile, while attack at the C of the cyclic species is unfavorable. The roles of the unique ligand set and low-spin nature of the NHase active site in function are also explored. It is found that the oxidized thiolate ligands are crucial to maintaining the LS state, which is important in the binding and activation of nitrile susbtrates. The dominant role of the backbone amidate ligands appears to be as a chelate in keeping the sulfenate properly oriented for nucleophilic attack on the coordinated substrate.

9.
Proteins ; 83(12): 2230-9, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26422370

RESUMEN

Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2-diol. Until now, six different H-Lyases have been studied. These H-Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N-1074 has two different isozymes of H-Lyase, HheA (A-type) and HheB (B-type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H-Lyases. Among the B-type H-Lyases, HheB shows relatively high enantioselectivity compared to that of HheBGP1 . This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3-dicyano-2-propanol at the catalytic site in the crystal structure of the HheB-DiCN complex suggests that the product should be (R)-epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity.


Asunto(s)
Corynebacterium/enzimología , Liasas/química , Liasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Epiclorhidrina/metabolismo , Liasas/genética , Modelos Moleculares , Nitrilos/química , Nitrilos/metabolismo , Propanoles/química , Propanoles/metabolismo , Conformación Proteica , Estereoisomerismo
10.
Angew Chem Int Ed Engl ; 54(37): 10763-7, 2015 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-26333053

RESUMEN

The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which ßArg56, strictly conserved and hydrogen bonded to the two post-translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2-1.3 Å). In combination with FTIR analyses of NHase following hydration in H2 (18) O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO(-) ) atom of αCys114-SO(-) , followed by nucleophilic attack of the S(SO(-) ) atom by a ßArg56-activated water molecule to release the product amide and regenerate αCys114-SO(-) .


Asunto(s)
Cristalografía por Rayos X/métodos , Cisteína/análogos & derivados , Hidroliasas/química , Ácidos Sulfénicos/química , Catálisis , Cisteína/química , Ligandos , Modelos Moleculares , Conformación Proteica
11.
J Biosci Bioeng ; 116(1): 22-7, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23453853

RESUMEN

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt (Co)-containing enzyme that catalyzes the hydrolysis of thiocyanate (SCN⁻), a major component of wastewater from coke oven factories, to carbonyl sulfide and ammonia. Although SCNase exhibits high structural similarities to Co-type nitrile hydratase (NHase), including a unique Co³âº catalytic center with two oxidized Cys ligands, both SCNase and NHase exclusively catalyze only their own substrates. Based on the differences in the substrate-binding pockets of these enzymes, ßArg90 and γArg136 of SCNase, with side chains extending toward the pocket, were separately substituted with Phe and Trp, the corresponding residues, respectively, in Co-type NHase. Both SCNase ßArg90 and SCNase γArg136 mutants showed no SCN⁻ hydrolysis activity but did catalyze the hydration of nitriles. The estimated kcat values (∼2 s⁻¹) corresponded to approximately 0.2% of that of Co-type NHase for nitrile hydration and approximately 3% of that of wild-type SCNase for SCN⁻ hydrolysis. The crystal structure of SCNase γR136W is essentially identical to that of the wild-type, including the Co³âº center having Cys oxidations; the size of the substrate pocket was enlarged because of conformational changes on the side chains of the mutated residue. Discussion of the difference in the environments around the substrate-binding pockets among the wild-type and mutant SCNases and Co-type NHase strongly suggests that ßArg90 and γArg136, positioned at the top of the Co³âº center, predominantly control the substrate selectivity of SCNase.


Asunto(s)
Arginina/química , Hidrolasas/química , Sustitución de Aminoácidos , Biocatálisis , Cobalto/química , Cisteína/química , Hidroliasas/química , Hidrolasas/genética , Modelos Moleculares , Oxidación-Reducción , Especificidad por Sustrato , Thiobacillus/enzimología
12.
Dalton Trans ; 42(9): 3092-9, 2013 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-23160436

RESUMEN

L-Cysteine desulfurase IscS and scaffold IscU proteins are universally involved in Fe/S cluster synthesis. The Archaeoglobus fulgidus (Af) genome encodes proteins having a high degree of primary structure similarity to IscS and IscU from other organisms. However, AfIscS is unusual because it lacks the active site lysine residue that normally forms an internal Schiff base with pyridoxal-phosphate (PLP) and serves as a base during catalysis. Our as-isolated recombinant AfIscS contains pyridoxamine phosphate (PMP) instead of the expected PLP and lacks desulfurase activity. We have solved its structure to 1.43 Å resolution and found that PMP binds non-covalently at the PLP site of the enzyme and displays significant disorder. However, the previously reported structure of recombinant Af(IscU-D35A-IscS)(2) contains an in vivo generated [Fe(2)S(2)] species within AfIscU and the question arises as to how its sulfides were generated. Here, we report that adding PLP to AfIscS produces an enzyme that displays in vitro L-cysteine desulfurase activity mediating the synthesis of a stable holo Af(IscU-D35A-IscS) complex.


Asunto(s)
Archaeoglobus fulgidus/enzimología , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Fosfato de Piridoxal/metabolismo
13.
PLoS One ; 6(8): e23716, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21858212

RESUMEN

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported. METHODOLOGY/PRINCIPAL FINDINGS: MTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the ß(8)α(8) barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions. CONCLUSIONS/SIGNIFICANCE: The different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.


Asunto(s)
Proteínas Bacterianas/química , Metilenotetrahidrofolato Reductasa (NADPH2)/química , Estructura Cuaternaria de Proteína , Thermus thermophilus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Biocatálisis , Cristalografía por Rayos X , Estabilidad de Enzimas , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura , Tetrahidrofolatos/química , Tetrahidrofolatos/metabolismo , Thermus thermophilus/genética
14.
J Biol Inorg Chem ; 15(5): 655-65, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20221653

RESUMEN

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe(3+) or Co(3+) center with two modified Cys ligands: cysteine sulfininate (Cys-SO(2) (-)) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, alphaS113A and betaY72F. The alphaS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV-vis absorption spectra indicated that the electronic state of the Fe center was altered by the alphaS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the alphaS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV-vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The betaY72F mutant exhibited no activity. The structure of the betaY72F mutant was highly conserved but was found to be the inactivated state, with alphaCys114-SO(H) oxidized to Cys-SO(2) (-), suggesting that betaTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.


Asunto(s)
Hidroliasas/química , Serina/química , Tirosina/química , Biocatálisis , Hidroliasas/metabolismo , Cinética , Ligandos , Modelos Moleculares , Mutación , Conformación Proteica , Rhodococcus/enzimología , Espectrofotometría Ultravioleta
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