RESUMEN
Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.
Asunto(s)
GMP-Reductasa/metabolismo , IMP Deshidrogenasa/metabolismo , Biocatálisis , Cristalografía por Rayos X , GMP-Reductasa/química , Guanosina Monofosfato/biosíntesis , Guanosina Monofosfato/química , Guanosina Monofosfato/metabolismo , Humanos , IMP Deshidrogenasa/química , Inosina Monofosfato/química , Inosina Monofosfato/metabolismo , Cinética , Modelos Moleculares , Estructura Molecular , NADP/química , NADP/metabolismo , Teoría Cuántica , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Human purine de novo synthesis pathway contains several multi-functional enzymes, one of which, tri-functional GART, contains three enzymatic activities in a single polypeptide chain. We have solved structures of two domains bearing separate catalytic functions: glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. Structures are compared with those of homologous enzymes from prokaryotes and analyzed in terms of the catalytic mechanism. We also report small angle X-ray scattering models for the full-length protein. These models are consistent with the enzyme forming a dimer through the middle domain. The protein has an approximate seesaw geometry where terminal enzyme units display high mobility owing to flexible linker segments. This resilient seesaw shape may facilitate internal substrate/product transfer or forwarding to other enzymes in the pathway.
Asunto(s)
Ligasas de Carbono-Nitrógeno/química , Fosforribosilglicinamida-Formiltransferasa/química , Adenosina Trifosfato/química , Sitios de Unión , Dominio Catalítico , Cristalografía , Glicina/química , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Ribosamonofosfatos/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.
Asunto(s)
Cristalización/métodos , Cristalografía/métodos , Péptido Hidrolasas/química , Proteínas/química , Proteínas/ultraestructura , Conformación ProteicaRESUMEN
The PP2A serine/threonine phosphatase regulates a plethora of cellular processes. In the cell the predominant form of the enzyme is a heterotrimer, formed by a core dimer composed of a catalytic and a scaffolding subunit, which assemble together with one of a range of different regulatory B subunits. Here, we present the first structure of a free non-complexed B subunit, B56 gamma. Comparison with the recent structures of a heterotrimeric complex and the core dimer reveals several significant conformational changes in the interface region between the B56 gamma and the core dimer. These allow for an assembly scheme of the PP2A holoenzyme to be put forth where B56 gamma first complexes with the scaffolding subunit and subsequently binds to the catalytic subunit and this induces the formation of a binding site for the invariant C-terminus of the catalytic subunit that locks in the complex as a last step of assembly.
Asunto(s)
Proteína Fosfatasa 2/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Escherichia coli/genética , Holoenzimas/química , Datos de Secuencia Molecular , Conformación Proteica , Multimerización de Proteína , Proteína Fosfatasa 2/aislamiento & purificación , Subunidades de Proteína/química , Alineación de SecuenciaRESUMEN
The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/química , Proteína Inhibidora de la Apoptosis Neuronal/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Cristalografía por Rayos X , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Modelos Moleculares , Datos de Secuencia Molecular , Proteína Inhibidora de la Apoptosis Neuronal/genética , Estructura Terciaria de Proteína/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ubiquitina-Proteína LigasasRESUMEN
Evasion of apoptosis is recognized as a characteristic of malignant growth. Anti-apoptotic B-cell lymphoma-2 (Bcl-2) family members have therefore emerged as potential therapeutic targets due to their critical role in proliferating cancer cells. Here, we present the crystal structure of Bfl-1, the last anti-apoptotic Bcl-2 family member to be structurally characterized, in complex with a peptide corresponding to the BH3 region of the pro-apoptotic protein Bim. The structure reveals distinct features at the peptide-binding site, likely to define the binding specificity for pro-apoptotic proteins. Superposition of the Bfl-1:Bim complex with that of Mcl-1:Bim reveals a significant local plasticity of hydrophobic interactions contributed by the Bim peptide, likely to be the basis for the multi specificity of Bim for anti-apoptotic proteins.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas de la Membrana/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Apoptosis , Proteína 11 Similar a Bcl2 , Cristalografía por Rayos X , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Estructura Secundaria de ProteínaRESUMEN
The implementation of generic and efficient technologies for the production of recombinant eukaryotic proteins remains an outstanding challenge in structural genomics programs. We have recently developed a new method for rapid identification of soluble protein expression in E. coli, the colony filtration blot (CoFi blot). In this study, the CoFi blot was used to screen libraries where the N-terminal translation start point was randomized. To investigate the efficiency of this strategy, we have attributed a large number of proteins to this process. In a set of 32 mammalian proteins, we were able to double the success rate (from 34 to 68%) of producing soluble and readily purifiable proteins in E. coli. Most of the selected constructs had their N-termini close to predicted domain borders and the method therefore provides a mean for experimental "domain foot printing." Surprisingly, for most of the targets, we also observed expressing constructs that were close to full-length. In summary this strategy constitutes a generic and efficient method for producing mammalian proteins for structural and functional studies.
Asunto(s)
Escherichia coli/metabolismo , Biblioteca de Genes , Proteínas Recombinantes/metabolismo , Clonación Molecular , Escherichia coli/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
Cytidine triphosphate synthetase (CTPS) is a key enzyme in nucleic acid and phospholipid biosynthesis and its activity is increased in certain human cancers, making it a promising drug target. The crystal structure of the synthetase domain of human CTPS, which represents the first structure of a CTPS from an eukaryote, has been determined. The structure is homotetrameric and each active site is formed by three different subunits. Sulfate ions bound to the active sites indicate the positions of phosphate-binding sites for the substrates ATP and UTP and the feedback inhibitor CTP. Together with earlier structures of bacterial CTPS, the human CTPS structure provides an extended understanding of the structure-function relationship of CTPS-family members. The structure also serves as a basis for structure-based design of anti-proliferative inhibitors.
Asunto(s)
Antineoplásicos , Ligasas de Carbono-Nitrógeno/química , Secuencia de Aminoácidos , Sitios de Unión , Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/aislamiento & purificación , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/enzimología , Retroalimentación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/químicaRESUMEN
Human NUDT16 is a member of the NUDIX hydrolase superfamily. After having been initially described as an mRNA decapping enzyme, recent studies conferred it a role as an "housecleaning" enzyme specialized in the removal of hazardous (deoxy)inosine diphosphate from the nucleotide pool. Here we present the crystal structure of human NUDT16 both in its apo-form and in complex with its product inosine monophosphate (IMP). NUDT16 appears as a dimer whose formation generates a positively charged trench to accommodate substrate-binding. Complementation of the structural data with detailed enzymatic and biophysical studies revealed the determinants of substrate recognition and particularly the importance of the substituents in position 2 and 6 on the purine ring. The affinity for the IMP product, harboring a carbonyl in position 6 on the base, compared to purine monophosphates lacking a H-bond acceptor in this position, implies a catalytic cycle whose rate is primarily regulated by the product-release step. Finally, we have also characterized a phenomenon of inhibition by the product of the reaction, IMP, which might exclude non-deleterious nucleotides from NUDT16-mediated hydrolysis regardless of their cellular concentration. Taken together, this study details structural and regulatory mechanisms explaining how substrates are selected for hydrolysis by human NUDT16.
Asunto(s)
Inosina Monofosfato/metabolismo , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Adenosina Difosfato/metabolismo , Biocatálisis/efectos de los fármacos , Calorimetría , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Inosina Monofosfato/farmacología , Cinética , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacosRESUMEN
SHIP2, OCRL, and INPP5B belong to inositol polyphosphate 5-phophatase subfamilies involved in insulin regulation and Lowes syndrome. The structural basis for membrane recognition, substrate specificity, and regulation of inositol polyphosphate 5-phophatases is still poorly understood. We determined the crystal structures of human SHIP2, OCRL, and INPP5B, the latter in complex with phosphoinositide substrate analogs, which revealed a membrane interaction patch likely to assist in sequestering substrates from the lipid bilayer. Residues recognizing the 1-phosphate of the substrates are highly conserved among human family members, suggesting similar substrate binding modes. However, 3- and 4-phosphate recognition varies and determines individual substrate specificity profiles. The high conservation of the environment of the scissile 5-phosphate suggests a common reaction geometry for all members of the human 5-phosphatase family.
Asunto(s)
Membrana Celular/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Modelos Moleculares , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Especificidad por SustratoAsunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Secuencia Conservada , Multimerización de Proteína , Pirofosfatasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Propiedades de Superficie , Hidrolasas NudixRESUMEN
Guanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors.
Asunto(s)
Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Multimerización de Proteína , Secuencia de Aminoácidos , Escherichia coli/química , Escherichia coli/enzimología , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Especificidad por SustratoRESUMEN
To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of "off target effects." However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔT(agg) around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.
Asunto(s)
Nucleósidos/metabolismo , Nucleótidos/metabolismo , Diseño de Fármacos , Pruebas de Enzimas/métodos , Enzimas/metabolismo , Guanina Desaminasa/química , Guanina Desaminasa/metabolismo , Humanos , Cinética , Redes y Vías Metabólicas , Nucleósidos/química , Nucleótidos/química , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/metabolismo , Uridina Fosforilasa/metabolismoRESUMEN
Ribonucleotide reductase (RR) is an α(n)ß(n) (RR1-RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP-GDP, TTP-ATP, and TTP-dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1-dATP hexamer and used single-particle electron microscopy to visualize the α(6)-ßß'-dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.
Asunto(s)
Dominio Catalítico , Nucleótidos/metabolismo , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/metabolismo , Saccharomyces cerevisiae/enzimología , Regulación Alostérica , Cristalografía por Rayos X , Nucleótidos de Desoxiadenina/química , Nucleótidos de Desoxiadenina/metabolismo , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Nucleótidos/química , Multimerización de Proteína , Ribonucleótido Reductasas/genética , Saccharomyces cerevisiae/químicaRESUMEN
UNLABELLED: The human SnoN is an oncoprotein that interacts with several transcription-regulatory proteins such as the histone-deacetylase, N-CoR containing co-repressor complex and Smad proteins. This study presents the crystal structure of the Dachshund homology domain of human SnoN. The structure reveals a groove composed of conserved residues with characteristic properties of a protein-interaction surface. A comparison of the 12 monomers in the asymmetric unit reveals the presence of two major conformations: an open conformation with a well accessible groove and a tight conformation with a less accessible groove. The variability in the backbone between the open and the tight conformations matches the differences seen in previously determined structures of individual Dachshund homology domains, suggesting a general plasticity within this fold family. The flexibility observed in the putative protein binding groove may enable SnoN to recognize multiple interaction partners. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Alineación de SecuenciaRESUMEN
Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch-Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (k(cat)/K(m)) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Guanina/metabolismo , Guanosina Monofosfato/metabolismo , Humanos , Hipoxantina/metabolismo , Hipoxantina Fosforribosiltransferasa/genética , Técnicas In Vitro , Cinética , Metaboloma , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
The Toll/interleukin-1 receptor (TIR) domain is a highly conserved signaling domain found in the intracellular regions of Toll-like receptors (TLRs), in interleukin-1 receptors, and in several cytoplasmic adaptor proteins. TIR domains mediate receptor signal transduction through recruitment of adaptor proteins and play critical roles in the innate immune response and inflammation. This work presents the 2.2A crystal structure of the TIR domain of human TLR10, revealing a symmetric dimer in the asymmetric unit. The dimer interaction surface contains residues from the BB-loop, DD-loop, and alphaC-helix, which have previously been identified as important structural motifs for signaling in homologous TLR receptors. The interaction surface is extensive, containing a central hydrophobic patch surrounded by polar residues. The BB-loop forms a tight interaction, where a range of consecutive residues binds in a pocket formed by the reciprocal BB-loop and alphaC-helix. This pocket appears to be well suited for binding peptide substrates, which is consistent with the notion that peptides and peptide mimetics of the BB-loop are inhibitors for TLR signaling. The TLR10 structure is in good agreement with available biochemical data on TLR receptors and is likely to provide a good model for the physiological dimer.
Asunto(s)
Citoplasma/química , Transducción de Señal , Receptor Toll-Like 10/química , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Mutación/genética , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Cytosolic 5'-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adenine-based nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5'-nucleotidase II as native protein (2.2 Angstrom) and in complex with adenosine (1.5 Angstrom) and beryllium trifluoride (2.15 Angstrom) The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5'(3')-deoxyribonucleotidase and cytosolic 5'-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5'-nucleotidase II with that of mitochondrial 5'(3')-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition.
Asunto(s)
5'-Nucleotidasa/química , Isoenzimas/química , Estructura Cuaternaria de Proteína , 2,3-Difosfoglicerato/química , 2,3-Difosfoglicerato/metabolismo , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Adenosina/química , Adenosina/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Axonal growth cone guidance is a central process in nervous system development and repair. Collapsin response mediator protein 2 (CRMP-2) is a neurite extension-promoting neuronal cytosolic molecule involved in the signalling of growth inhibitory cues from external stimuli, such as semaphorin 3A and the myelin-associated glycoprotein. We have determined the crystal structure of human tetrameric CRMP-2, which is structurally related to the dihydropyriminidases; however, the active site is not conserved. The wealth of earlier functional mapping data for CRMP-2 are discussed in light of the three-dimensional structure of the protein. The differences in oligomerisation interfaces between CRMP-1 and CRMP-2 are used to model CRMP-1/2 heterotetramers.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/química , Proteínas del Tejido Nervioso/química , Animales , Clonación Molecular/métodos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Modelos Moleculares , Proteínas del Tejido Nervioso/genética , Biosíntesis de Proteínas/fisiologíaRESUMEN
Inosine triphosphatase (ITPA) is a ubiquitous key regulator of cellular non-canonical nucleotide levels. It breaks down inosine and xanthine nucleotides generated by deamination of purine bases. Its enzymatic action prevents accumulation of ITP and reduces the risk of incorporation of potentially mutagenic inosine nucleotides into nucleic acids. Here we describe the crystal structure of human ITPA in complex with its prime substrate ITP, as well as the apoenzyme at 2.8 and 1.1A, respectively. These structures show for the first time the site of substrate and Mg2+ coordination as well as the conformational changes accompanying substrate binding in this class of enzymes. Enzyme substrate interactions induce an extensive closure of the nucleotide binding grove, resulting in tight interactions with the base that explain the high substrate specificity of ITPA for inosine and xanthine over the canonical nucleotides. One of the dimer contact sites is made up by a loop that is involved in coordinating the metal ion in the active site. We predict that the ITPA deficiency mutation P32T leads to a shift of this loop that results in a disturbed affinity for nucleotides and/or a reduced catalytic activity in both monomers of the physiological dimer.