RESUMEN
Arrestin-1 is the arrestin family member responsible for inactivation of the G protein-coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1-enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.
Asunto(s)
Arrestina/química , Biomarcadores de Tumor/química , Proteínas de Unión al ADN/química , Modelos Moleculares , Complejos Multienzimáticos/química , Fosfopiruvato Hidratasa/química , Rodopsina/química , Proteínas Supresoras de Tumor/química , Arrestina/genética , Sitios de Unión , Biomarcadores de Tumor/genética , Catálisis , Proteínas de Unión al ADN/genética , Humanos , Complejos Multienzimáticos/genética , Fosfopiruvato Hidratasa/genética , Rodopsina/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.
Asunto(s)
Basigina/metabolismo , Anhidrasa Carbónica IV/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Mapas de Interacción de Proteínas , Secuencia de Aminoácidos , Animales , Basigina/química , Células HEK293 , Humanos , Proteínas de la Membrana , Modelos Moleculares , Dominios Proteicos , Ratas , Alineación de Secuencia , Simportadores/metabolismo , XenopusRESUMEN
Asparagine synthetase (ASNS) converts aspartate and glutamine to asparagine and glutamate in an ATP-dependent reaction. ASNS is present in most, if not all, mammalian organs, but varies widely in basal expression. Human ASNS activity is highly responsive to cellular stress, primarily by increased transcription from a single gene located on chromosome 7. Elevated ASNS protein expression is associated with resistance to asparaginase therapy in childhood acute lymphoblastic leukemia. There is evidence that ASNS expression levels may also be inversely correlated with asparaginase efficacy in certain solid tumors as well. Children with mutations in the ASNS gene exhibit developmental delays, intellectual disability, microcephaly, intractable seizures, and progressive brain atrophy. Thus far, 15 unique mutations in the ASNS gene have been clinically associated with asparagine synthetase deficiency (ASD). Molecular modeling using the Escherichia coli ASNS-B structure has revealed that most of the reported ASD substitutions are located near catalytic sites or within highly conserved regions of the protein. For some ASD patients, fibroblast cell culture studies have eliminated protein and mRNA synthesis or stability as the basis for decreased proliferation.
Asunto(s)
Aspartatoamoníaco Ligasa/genética , Regulación Enzimológica de la Expresión Génica , Mutación , Animales , Asparaginasa/uso terapéutico , Aspartatoamoníaco Ligasa/química , Aspartatoamoníaco Ligasa/fisiología , Resistencia a Medicamentos/genética , Predisposición Genética a la Enfermedad , HumanosRESUMEN
The combination of X-ray free-electron lasers (XFELs) with serial femtosecond crystallography represents cutting-edge technology in structural biology, allowing the study of enzyme reactions and dynamics in real time through the generation of `molecular movies'. This technology combines short and precise high-energy X-ray exposure to a stream of protein microcrystals. Here, the XFEL structure of carbonic anhydrase II, a ubiquitous enzyme responsible for the interconversion of CO2 and bicarbonate, is reported, and is compared with previously reported NMR and synchrotron X-ray and neutron single-crystal structures.
Asunto(s)
Anhidrasa Carbónica II , Anhidrasa Carbónica II/química , Cristalografía por Rayos X , Proteínas/química , Sincrotrones , Rayos X , HumanosRESUMEN
[This corrects the article DOI: 10.1107/S2052252520000986.].
RESUMEN
Here we report the synthesis of a series of taurine substituted sulfonamide derivatives 1-29 having the ureido moiety installed at the tail section as selective inhibitors of the tumor associated human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) IX and XII. The series was deeply investigated for their kinetic features which demonstrated a strong dependence on the ureido moiety. High resolution X-ray crystallographic investigation on selected ligand adducts complexed with hCA II and hCA IX-mimic revealed a strong correlation between the ureido moiety and the amino acid residues Q92 and Q67 in both the hCA II and hCA IX-mimic, contributing to highly stabilized ligand-protein complex.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Nitric oxide (NO) promotes vasodilation through the activation of guanylate cyclase, resulting in the relaxation of the smooth muscle vasculature and a subsequent decrease in blood pressure. Therefore, its regulation is of interest for the treatment and prevention of heart disease. An example is pulmonary hypertension which is treated by targeting this NO/vasodilation pathway. In bacteria, plants and fungi, nitrite (NO2 -) is utilized as a source of NO through enzymes known as nitrite reductases. These enzymes reduce NO2 - to NO through a catalytic metal ion, often copper. Recently, several studies have shown nitrite reductase activity of mammalian carbonic anhydrase II (CAII), yet the molecular basis for this activity is unknown. Here we report the crystal structure of copper-bound human CAII (Cu-CAII) in complex with NO2 - at 1.2â Å resolution. The structure exhibits Type 1 (T-1) and 2 (T-2) copper centers, analogous to bacterial nitrite reductases, both required for catalysis. The copper-substituted CAII active site is penta-coordinated with a 'side-on' bound NO2 -, resembling a T-2 center. At the N terminus, several residues that are normally disordered form a porphyrin ring-like configuration surrounding a second copper, acting as a T-1 center. A structural comparison with both apo- (without metal) and zinc-bound CAII (Zn-CAII) provides a mechanistic picture of how, in the presence of copper, CAII, with minimal conformational changes, can function as a nitrite reductase.
RESUMEN
The carbonic anhydrases (CAs; EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide (CO2) and bicarbonate (HCO3-). Since their discovery in 1933, CAs have been at the forefront of scientific discovery: the understanding of enzymatic reactions, structural biology, molecular dynamics, drug discovery, and clinical medicine. These ubiquitous enzymes equilibrate the reaction between CO2, HCO3-, and protons. Hence, CAs have important roles in ion transport, acid-base regulation, gas exchange, photosynthesis, and CO2 fixation. In this chapter, we describe the protocols leading to, and the analysis of CA neutron crystal structures. This accumulation of structural knowledge adds to our understanding of the enzymatic mechanism and development of CA inhibitors.
Asunto(s)
Anhidrasas Carbónicas , Bicarbonatos , Dióxido de Carbono , Inhibidores de Anhidrasa Carbónica , NeutronesRESUMEN
Tumor cells rely on glycolysis to meet their elevated demand for energy. Thereby they produce significant amounts of lactate and protons, which are exported via monocarboxylate transporters (MCTs), supporting the formation of an acidic microenvironment. The present study demonstrates that carbonic anhydrase IX (CAIX), one of the major acid/base regulators in cancer cells, forms a protein complex with MCT1 and MCT4 in tissue samples from human breast cancer patients, but not healthy breast tissue. Formation of this transport metabolon requires binding of CAIX to the Ig1 domain of the MCT1/4 chaperon CD147 and is required for CAIX-mediated facilitation of MCT1/4 activity. Application of an antibody, directed against the CD147-Ig1 domain, displaces CAIX from the transporter and suppresses CAIX-mediated facilitation of proton-coupled lactate transport. In cancer cells, this "metabolon disruption" results in a decrease in lactate transport, reduced glycolysis, and ultimately reduced cell proliferation. Taken together, the study shows that carbonic anhydrases form transport metabolons with acid/base transporters in human tumor tissue and that these interactions can be exploited to interfere with tumor metabolism and proliferation.
Asunto(s)
Neoplasias de la Mama/patología , Anhidrasa Carbónica IX/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Simportadores/metabolismo , Basigina/química , Basigina/metabolismo , Humanos , Células MCF-7 , Modelos Moleculares , Dominios ProteicosRESUMEN
Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3 -) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (k cat and k cat/K m) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure-function relationships of many other biologically important enzymes.
RESUMEN
This study provides a structure-activity relationship study of a series of lipophilic carbonic anhydrase (CA) inhibitors with an acetazolamide backbone. The inhibitors were tested against the tumor-expressed CA isozyme IX (CA IX), and the cytosolic CA I, CA II, and membrane-bound CA IV. The study identified several low nanomolar potent inhibitors against CA IX, with lipophilicities spanning two log units. Very potent pan-inhibitors with nanomolar potency against CA IX and sub-nanomolar potency against CA II and CA IV, and with potency against CA I one order of magnitude better than the parent acetazolamide 1 were also identified in this study, together with compounds that displayed selectivity against membrane-bound CA IV. A comprehensive X-ray crystallographic study (12 crystal structures), involving both CA II and a soluble CA IX mimetic (CA IX-mimic), revealed the structural basis of this particular inhibition profile and laid the foundation for further developments toward more potent and selective inhibitors for the tumor-expressed CA IX.
Asunto(s)
Acetazolamida/química , Anhidrasa Carbónica IX/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Acetazolamida/metabolismo , Sitios de Unión , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasa Carbónica IX/genética , Inhibidores de Anhidrasa Carbónica/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Simulación de Dinámica Molecular , Neoplasias/enzimología , Neoplasias/patología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
Why metalloenzymes often show dramatic changes in their catalytic activity when subjected to chemically similar but non-native metal substitutions is a long-standing puzzle. Here, we report on the catalytic roles of metal ions in a model metalloenzyme system, human carbonic anhydrase II (CA II). Through a comparative study on the intermediate states of the zinc-bound native CA II and non-native metal-substituted CA IIs, we demonstrate that the characteristic metal ion coordination geometries (tetrahedral for Zn2+, tetrahedral to octahedral conversion for Co2+, octahedral for Ni2+, and trigonal bipyramidal for Cu2+) directly modulate the catalytic efficacy. In addition, we reveal that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring water network in the active site. Our study provides evidence that the metal ions in metalloenzymes have a crucial impact on the catalytic mechanism beyond their primary chemical properties.
Asunto(s)
Anhidrasas Carbónicas/química , Iones/química , Metaloproteínas/química , Metales/química , Sitios de Unión , Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/metabolismo , Anhidrasas Carbónicas/metabolismo , Catálisis , Dominio Catalítico , Cobalto/química , Cobre/química , Cristalografía por Rayos X , Humanos , Iones/metabolismo , Cinética , Metaloproteínas/metabolismo , Metales/metabolismo , Modelos Moleculares , Níquel/química , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Zinc/químicaRESUMEN
Cell surface-localized P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans mediates sucrose-independent adhesion to tooth surfaces. Previous studies showed that P1's C-terminal segment (C123, AgII) is also liberated as a separate polypeptide, contributes to cellular adhesion, interacts specifically with intact P1 on the cell surface, and forms amyloid fibrils. Identifying how C123 specifically interacts with P1 at the atomic level is essential for understanding related virulence properties of S. mutans. However, with sizes of ~ 51 and ~ 185 kDa, respectively, C123 and full-length P1 are too large to achieve high-resolution data for full structural analysis by NMR. Here, we report on biologically relevant interactions of the individual C3 domain with A3VP1, a polypeptide that represents the apical head of P1 as it is projected on the cell surface. Also evaluated are C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15 N-enriched C3 demonstrate its specific binding to A3VP1. Based on resolved C3 assignments, two binding sites, proximal and distal, are identified. Complementary NMR titration of A3VP1 with a C3/C12 complex suggests that binding of A3VP1 occurs on the distal C3 binding site, while the proximal site is occupied by C12. The MAb 6-8C binding interface to C3 overlaps with that of A3VP1 at the distal site. Together, these results identify a specific C3-A3VP1 interaction that serves as a foundation for understanding the interaction of C123 with P1 on the bacterial surface and the related biological processes that stem from this interaction. DATABASE: BMRB submission code: 27935.
Asunto(s)
Adhesinas Bacterianas/química , Resonancia Magnética Nuclear Biomolecular , Streptococcus mutans/química , Cristalografía por Rayos X , Modelos Moleculares , Unión ProteicaRESUMEN
3,17ß-Bis-sulfamoyloxy-2-methoxyestra-1,3,5(10)-triene (STX140), a bis-sulfamate derivative of the endogenous steroid 2-methoxyestradiol, has shown promising anticancer potency both in vitro and in vivo, with excellent bioavailability. Its activity against taxane-resistant xenografts makes it a potential drug candidate against triple-negative breast cancer (TNBC). These properties are linked to the ability of STX140 to act in a multitargeting fashion in vivo as a microtubule disruptor, leading to cell cycle arrest and with both proapoptotic and anti-angiogenic activities. Carbonic anhydrase IX (CA IX) is a well-established biomarker for aggressive cancers, including TNBC. This study reports, for the first time, the inhibitory activities of a series of steroidal and nonsteroidal sulfamate derivatives against CA IX in comparison to the ubiquitous CA II, with some compounds demonstrating 100-200-fold selectivity for CA IX over CA II. X-ray crystallographic studies of four of the most promising compounds reveal that isoform-specific residue interactions are responsible for the high specificity.
Asunto(s)
Antígenos de Neoplasias/química , Antineoplásicos/química , Anhidrasa Carbónica IX/química , Inhibidores de Anhidrasa Carbónica/química , Estrenos/química , Antígenos de Neoplasias/metabolismo , Antineoplásicos/metabolismo , Anhidrasa Carbónica IX/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Estrenos/metabolismo , Humanos , Cinética , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
X-ray and neutron crystallography are powerful techniques utilized to study the structures of biomolecules. Visualization of enzymes in complex with substrate/product and the capture of intermediate states can be related to activity to facilitate understanding of the catalytic mechanism. Subsequent analysis of small molecule binding within the enzyme active site provides insight into mechanisms of inhibition, supporting the design of novel inhibitors using a structure-guided approach. The first X-ray crystal structures were determined for small, ubiquitous enzymes such as carbonic anhydrase (CA). CAs are a family of zinc metalloenzymes that catalyze the hydration of CO2, producing HCO3 - and a proton. The CA structure and ping-pong mechanism have been extensively studied and are well understood. Though the function of CA plays an important role in a variety of physiological functions, CA has also been associated with diseases such as glaucoma, edema, epilepsy, obesity, and cancer and is therefore recognized as a drug target. In this review, a brief history of crystallography and its impact on CA research is discussed.
RESUMEN
Carbonic anhydrase II (CA II) is a zinc metalloenzyme that catalyzes the reversible interconversion of water and CO2 to bicarbonate and a proton. CA II is abundant in most cells, and plays a role in numerous processes including gas exchange, epithelial ion transport, respiration, extra- and intracellular pH control, and vascular regulation. Beyond these CO2 and pH-linked roles, it has been postulated that CA II might also reduce nitrite (NO2-) to nitric oxide (NO), as bicarbonate and NO2- both exhibit sp2 molecular geometry and NO also plays an important role in vasodilation and regulation of blood pressure. Indeed, previous studies by Aamand et al. have shown that bovine CA II (BCA II) possesses nitrite dehydration activity and paradoxically demonstrated that CA inhibitors (CAIs) such as dorzolamide and acetazolamide significantly increased NO production (Aamand et al., 2009; Nielsen and Fago, 2015) [1,2]. Hence, the goal of this work was to revisit these studies using the same experimental conditions as Aamand et al. measuring NO generation by two methods, and to examine the structure of CA II in complex with NO2- in the presence and absence of dorzolamide. Our results contradict the previous findings and indicate that CA II does not exhibit nitrite reductase or dehydration activity, and that this is not enhanced in the presence of CA inhibitors. In addition, a structural examination of BCA II in complex with NO2- and superimposed with dorzolamide demonstrates that CA inhibitor binding at the active site to the zinc moiety blocks potential NO2- binding.
Asunto(s)
Anhidrasa Carbónica II/química , Nitrito Reductasas/química , Oxidorreductasas/química , Animales , Bovinos , Cristalografía por Rayos XRESUMEN
Recent advances in X-ray free-electron laser (XFEL) sources have permitted the study of protein dynamics. Femtosecond X-ray pulses have allowed the visualization of intermediate states in enzyme catalysis. In this study, the growth of carbonic anhydrase II microcrystals (40-80â µm in length) suitable for the collection of XFEL diffraction data at the Pohang Accelerator Laboratory is demonstrated. The crystals diffracted to 1.7â Å resolution and were indexed in space group P21, with unit-cell parameters a = 42.2, b = 41.2, c = 72.0â Å, ß = 104.2°. These preliminary results provide the necessary framework for time-resolved experiments to study carbonic anhydrase catalysis at XFEL beamlines.
Asunto(s)
Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/genética , Secuencia de Aminoácidos , Cristalización/métodos , Cristalografía por Rayos X/métodos , Rayos LáserRESUMEN
The combination of a ß-adrenergic receptors (AR) blocker and a carbonic anhydrase (CA, EC 4.2.1.1) inhibitor in eye drops formulations is one of the most clinically used treatment for glaucoma. A novel approach consisting of single-molecule, multitargeted compounds for the treatment of glaucoma is proposed here by designing compounds which concomitantly interact with the ß-adrenergic and CA targets. Most derivatives of the two series of benzenesulfonamides incorporating 2-hydroxypropylamine moieties reported here exhibited striking efficacy against the target hCA II and XII, whereas a subset of compounds also showed significant modulation of ß1- and ß2-ARs. X-ray crystallography studies provided rationale for the observed hCA inhibition. The best dual-agents decreased IOP more effectively than clinically used dorzolamide, timolol, and the combination of them in an animal model of glaucoma. The reported evidence supports the proof-of-concept of ß-ARs blocker-CAI hybrids for antiglaucoma therapy with an innovative mechanism of action.