RESUMEN
LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3beta-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3beta-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/metabolismo , Modelos Moleculares , Mutagénesis , Proteína Niemann-Pick C1 , Estructura Terciaria de ProteínaRESUMEN
Human plasma low-density lipoproteins (LDL), a risk factor for cardiovascular disease, transfer cholesterol from plasma to liver cells via the LDL receptor (LDLr). Here, we report the structures of LDL and its complex with the LDL receptor extracellular domain (LDL.LDLr) at extracellular pH determined by cryoEM. Difference imaging between LDL.LDLr and LDL localizes the site of LDLr bound to its ligand. The structural features revealed from the cryoEM map lead to a juxtaposed stacking model of cholesteryl esters (CEs). High density in the outer shell identifies protein-rich regions that can be accounted for by a single apolipoprotein (apo B-100, 500 kDa) leading to a model for the distribution of its alpha-helix and beta-sheet rich domains across the surface. The structural relationship between the apo B-100 and CEs appears to dictate the structural stability and function of normal LDL.
Asunto(s)
Microscopía por Crioelectrón/métodos , Modelos Moleculares , Receptores de LDL/química , Humanos , Conformación ProteicaRESUMEN
The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals an interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.
Asunto(s)
Anopheles/inmunología , Proteínas del Sistema Complemento/metabolismo , Proteínas de Insectos/metabolismo , Animales , Cristalografía por Rayos X , Cisteína/metabolismo , Hemolinfa/inmunología , Proteínas de Insectos/química , Proteínas de Insectos/genética , Conformación Proteica , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14's action is unknown. Here we present the crystal structure of S14 to 2.65 Å and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.
Asunto(s)
Ácidos Grasos/biosíntesis , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Ácidos Grasos/química , Ácidos Grasos/genética , Femenino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Factores de Transcripción/genéticaRESUMEN
Ushers constitute a family of bacterial outer membrane proteins responsible for the assembly and secretion of surface organelles such as the pilus. The structure at 3.15-A resolution of the usher pyelonephritis-associated pili C (PapC) translocation domain reveals a 24-stranded kidney-shaped beta-barrel, occluded by an internal plug domain. The dimension of the pore allows tandem passage of individual folded pilus subunits in an upright pilus growth orientation, but is insufficient for accommodating donor strand exchange. The molecular packing revealed by the crystal structure shows that 2 PapC molecules in head-to-head orientation interact via exposed beta-strand edges, which could be the preferred dimer interaction in solution. In vitro reconstitution of fiber assemblies suggest that PapC monomers may be sufficient for fiber assembly and secretion; both the plug domain and the C-terminal domain of PapC are required for filament assembly, whereas the N-terminal domain is mainly responsible for recruiting the chaperone-subunit complexes to the usher. The plug domain has a dual function: gating the beta-pore and participating in pilus assembly.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestructura , Fimbrias Bacterianas/química , Fimbrias Bacterianas/ultraestructura , Porinas/química , Porinas/ultraestructura , Cristalografía por Rayos X , Proteínas de Escherichia coli/genética , Porinas/genética , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Presentation of antigenic peptides to T-cell receptors is an essential step in the adaptive immune response. In the mouse the class Ib major histocompatibility complex molecule, H2-M3, presents bacterial- and mitochondrial-derived peptides to T-cell receptors on cytotoxic T cells. Four mitochondrial heptapeptides, differing only at residue 6, form complexes with H2-M3 which can be distinguished by T cells. No structures of relevant receptors are available. To investigate the structural basis for this distinction, crystal structures were determined and molecular dynamics simulations over one microsecond were done for each complex. In the crystal structures of the heptapeptide complexes with H2-M3, presented here, the side chains of the peptide residues at position 6 all point into the H2-M3 binding groove, and are thus inaccessible, so that the very similar structures do not suggest how recognition and initiation of responses by the T cells may occur. However, conformational differences, which could be crucial to T-cell discrimination, appear within one microsecond during molecular dynamics simulations of the four complexes. Specifically, the three C-terminal residues of peptide ligands with alanine or threonine at position 6 partially exit the binding groove; this does not occur in peptide ligands with isoleucine or valine at position 6. Structural changes associated with partial peptide exit from the binding groove, along with relevant peptide binding energetics and immunological results are discussed. Communicated by Ramaswamy H. Sarma.
Asunto(s)
Péptidos , Linfocitos T Citotóxicos , Ratones , Animales , Péptidos/química , Ligandos , Histocompatibilidad , Antígenos de Histocompatibilidad Clase I/químicaRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally regulates hepatic low-density lipoprotein receptors (LDLRs) by binding to LDLRs on the cell surface, leading to their degradation. The binding site of PCSK9 has been localized to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. Here, we describe the crystal structure of a complex between PCSK9 and the EGF-A domain of the LDLR. The binding site for the LDLR EGF-A domain resides on the surface of PCSK9's subtilisin-like catalytic domain containing Asp-374, a residue for which a gain-of-function mutation (Asp-374-Tyr) increases the affinity of PCSK9 toward LDLR and increases plasma LDL-cholesterol (LDL-C) levels in humans. The binding surface on PCSK9 is distant from its catalytic site, and the EGF-A domain makes no contact with either the C-terminal domain or the prodomain. Point mutations in PCSK9 that altered key residues contributing to EGF-A binding (Arg-194 and Phe-379) greatly diminished binding to the LDLR's extracellular domain. The structure of PCSK9 in complex with the LDLR EGF-A domain defines potential therapeutic target sites for blocking agents that could interfere with this interaction in vivo, thereby increasing LDLR function and reducing plasma LDL-C levels.
Asunto(s)
Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Sitios de Unión , Línea Celular , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Lipoproteínas LDL/sangre , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proproteína Convertasa 9 , Proproteína Convertasas , Unión Proteica , Conformación Proteica , Receptores de LDL/química , Serina Endopeptidasas/química , Serina Endopeptidasas/genéticaRESUMEN
The Drosophila peptidoglycan recognition protein SA (PGRP-SA) is critically involved in sensing bacterial infection and activating the Toll signaling pathway, which induces the expression of specific antimicrobial peptide genes. We have determined the crystal structure of PGRP-SA to 2.2-A resolution and analyzed its peptidoglycan (PG) recognition and signaling activities. We found an extended surface groove in the structure of PGRP-SA, lined with residues that are highly diverse among different PGRPs. Mutational analysis identified it as a PG docking groove required for Toll signaling and showed that residue Ser158 is essential for both PG binding and Toll activation. Contrary to the general belief that PGRP-SA has lost enzyme function and serves primarily for PG sensing, we found that it possesses an intrinsic L,D-carboxypeptidase activity for diaminopimelic acid-type tetrapeptide PG fragments but not lysine-type PG fragments, and that Ser158 and His42 may participate in the hydrolytic activity. As L,D-configured peptide bonds exist only in prokaryotes, this work reveals a rare enzymatic activity in a eukaryotic protein known for sensing bacteria and provides a possible explanation of how PGRP-SA mediates Toll activation specifically in response to lysine-type PG.
Asunto(s)
Carboxipeptidasas/química , Proteínas Portadoras/química , Peptidoglicano/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cromatografía Líquida de Alta Presión , Cruzamientos Genéticos , Cristalografía por Rayos X , Análisis Mutacional de ADN , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Hidrólisis , Lisina/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Receptores Toll-Like/metabolismo , Tirosina/químicaRESUMEN
Like pieces belonging to a large mosaic, the structures of low-density lipoprotein receptor (LDL-R) modules have been elucidated one by one in recent years. LDL-Rs localized on hepatocytes play an important role in removing cholesterol-transporting LDL particles from the plasma by receptor-mediated endocytosis. Key steps in this process involve the LDL-R binding LDL at neutral pH at the cell surface and, after internalization, releasing it again at acidic pH in the endosomes. How the modules of the LDL-R might interact within the intact receptor to carry out ligand binding and release has been revealed by the recent crystal structure of the extracellular domain of the LDL-R.
Asunto(s)
Endocitosis/fisiología , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/metabolismo , Hepatocitos/metabolismo , Lipoproteínas LDL/química , Lipoproteínas LDL/metabolismo , Receptores de LDL/química , Receptores de LDL/metabolismo , Animales , Sitios de Unión , Transporte Biológico Activo/fisiología , Endosomas/metabolismo , Humanos , Ligandos , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel beta-barrel traverses the outer membrane and adjacent beta-strands are connected by extracellular loops and periplasmic turns. Located inside the beta-barrel is the plug domain, composed primarily of a mixed four-stranded beta-sheet and a series of interspersed alpha-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Sideróforos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico Activo , Proteínas Portadoras/química , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Bacterias Gramnegativas/química , Hierro/química , Hierro/metabolismo , Ligandos , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Periplasma/metabolismo , Conformación Proteica , Receptores Virales/química , Sideróforos/química , Transducción de SeñalRESUMEN
We first describe the history and methods of membrane protein crystallization, and show how the structure of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas viridis was solved. The structure of this membrane protein complex is correlated with its function as a light-driven electron pump across the photosynthetic membrane. Finally we draw conclusions on the structure of the photosystem II reaction centre from plants and discuss the aspects of membrane protein structure.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética , Rhodopseudomonas/metabolismo , Rhodopseudomonas/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Bacteriorodopsinas/química , Evolución Biológica , Membrana Celular/metabolismo , Cristalografía , Citocromos/química , Detergentes/farmacología , Electrones , Membrana Dobles de Lípidos , Lípidos/química , Microscopía , Microscopía Electrónica , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Premio Nobel , Fotosíntesis , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas/química , Proteobacteria/fisiología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Agua/química , Difracción de Rayos XRESUMEN
BACKGROUND: NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels. PRINCIPAL FINDINGS: Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent. CONCLUSION: The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.
Asunto(s)
Proteínas de la Membrana/química , Secuencia de Aminoácidos , Colesterol/metabolismo , Cristalografía por Rayos X , Humanos , Glicoproteínas de Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
Thioester-containing proteins (TEPs) are a major component of the innate immune response of insects to invasion by bacteria and protozoa. TEPs form a distinct clade of a superfamily that includes the pan-protease inhibitors alpha(2)-macroglobulins and vertebrate complement factors. The essential feature of these proteins is a sequestered thioester bond that, after cleavage in a protease-sensitive region of the protein, is activated and covalently binds to its target. Recently, TEP1 from the malarial vector Anopheles gambiae was shown to mediate recognition and killing of ookinetes from the malarial parasite Plasmodium berghei, a model for the human malarial parasite Plasmodium falciparum. Here, we present the crystal structure of the TEP1 isoform TEP1r. Although the overall protein fold of TEP1r resembles that of complement factor C3, the TEP1r domains are repositioned to stabilize the inactive conformation of the molecule (containing an intact thioester) in the absence of the anaphylotoxin domain, a central component of complement factors. The structure of TEP1r provides a molecular basis for the differences between TEP1 alleles TEP1r and TEP1s, which correlate with resistance of A. gambiae to infection by P. berghei.
Asunto(s)
Anopheles/química , Complemento C3/química , Complemento C3/fisiología , Glicoproteínas/química , Glicoproteínas/fisiología , Proteínas de Insectos/química , Proteínas de Insectos/fisiología , Malaria/prevención & control , Plasmodium berghei/crecimiento & desarrollo , Homología Estructural de Proteína , Animales , Anopheles/inmunología , Anopheles/parasitología , Línea Celular , Secuencia Conservada , Cristalografía por Rayos X , Malaria/metabolismo , Malaria/parasitología , Plasmodium berghei/inmunología , Estructura Terciaria de ProteínaRESUMEN
Transcription of the ferric citrate import system is regulated by ferric citrate binding to the outer membrane transporter FecA. A signal indicating transporter occupancy is relayed across the outer membrane to energy-transducing and regulatory proteins embedded in the cytoplasmic membrane. Because transcriptional activation is not coupled to ferric citrate import, an allosteric mechanism underlies this complex signaling mechanism. Using evolution-based statistical analysis we have identified a sparse but structurally connected network of residues that links distant functional sites in FecA. Functional analyses of these positions confirm their involvement in the mechanism that regulates transcriptional activation in response to ferric citrate binding at the cell surface. This mechanism appears to be conserved and provides the structural basis for the allosteric signaling of TonB-dependent transporters.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico Activo , Fenómenos Biofísicos , Biofisica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de la Membrana/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Receptores de Superficie Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
Tracheal cytotoxin (TCT), a naturally occurring fragment of Gram-negative peptidoglycan, is a potent elicitor of innate immune responses in Drosophila. It induces the heterodimerization of its recognition receptors, the peptidoglycan recognition proteins (PGRPs) LCa and LCx, which activates the immune deficiency pathway. The crystal structure at 2.1 angstrom resolution of TCT in complex with the ectodomains of PGRP-LCa and PGRP-LCx shows that TCT is bound to and presented by the LCx ectodomain for recognition by the LCa ectodomain; the latter lacks a canonical peptidoglycan-docking groove conserved in other PGRPs. The interface, revealed in atomic detail, between TCT and the receptor complex highlights the importance of the anhydro-containing disaccharide in bridging the two ectodomains together and the critical role of diaminopimelic acid as the specificity determinant for PGRP interaction.
Asunto(s)
Proteínas Portadoras/química , Citotoxinas/química , Peptidoglicano/química , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Cristalización , Cristalografía por Rayos X , Citotoxinas/metabolismo , Drosophila melanogaster , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Peptidoglicano/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Cryptochromes use near-UV/blue light to regulate a variety of growth and adaptive process. Recent biochemical studies demonstrate that the Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (Cry-DASH) subfamily of cryptochromes have photolyase activity exclusively for single-stranded cyclobutane pyrimidine dimer (CPD)-containing DNA substrate [Selby C, Sancar A (2006) Proc Natl Acad Sci USA 103:17696-17700]. The crystal structure of cryptochrome 3 from Arabidopsis thaliana (At-Cry3), a member of the Cry-DASH proteins, at 2.1 A resolution, reveals that both the light-harvesting cofactor 5,10-methenyl-tetrahydrofolyl-polyglutamate (MTHF) and the catalytic cofactor flavin adenine dinucleotide (FAD) are noncovalently bound to the protein. The residues responsible for binding of MTHF in At-Cry3 are not conserved in Escherichia coli photolyase but are strongly conserved in the Cry-DASH subfamily of cryptochromes. The distance and orientation between MTHF and flavin adenine dinucleotide in At-Cry3 is similar to that of E. coli photolyase, in conjunction with the presence of electron transfer chain, suggesting the conservation of redox activity in At-Cry3. Two amino acid substitutions and the penetration of three charged side chains into the CPD-binding cavity in At-Cry3 alter the hydrophobic environment that is accommodating the hydrophobic sugar ring and thymine base moieties in class I CPD photolyases. These changes most likely make CPD binding less energetically favorable and, hence, insufficient to compete with pairing and stacking interactions between the CPD and the duplex DNA substrate. Thus, Cry-DASH subfamily proteins may be unable to stabilize CPD flipped out from the duplex DNA substrate but may be able to preserve the DNA repair activity toward single-stranded CPD-containing DNA substrate.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Desoxirribodipirimidina Fotoliasa/química , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Coenzimas , Criptocromos , Cristalografía por Rayos X , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Transporte de Electrón , Flavina-Adenina Dinucleótido/metabolismo , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Ácido Fólico/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The covalent attachment of heme cofactors to the apo-polypeptides via thioether bonds is unique to the maturation of c-type cytochromes. A number of thiol-disulfide oxidoreductases prepare the apocytochrome for heme insertion in system I and II cytochrome c maturation. Although most thiol-disulfide oxidoreductases are nonspecific, the less common, specific thiol-disulfide oxidoreductases may be key to directing the usage of electrons. Here we demonstrate that unlike other thiol-disulfide oxidoreductases, the protein responsible for reducing oxidized apocytochrome c in Bacillus subtilis, ResA, is specific for cytochrome c550 and utilizes alternate conformations to recognize redox partners. We report solution NMR evidence that ResA undergoes a redox-dependent conformational change between oxidation states, as well as data showing that ResA utilizes a surface cavity present only in the reduced state to recognize a peptide derived from cytochrome c550. Finally, we confirm that ResA is a specific thiol-disulfide oxidoreductase by comparing its reactivity to our mimetic peptide with its reactivity to oxidized glutathione, a nonspecific substrate. This study biochemically demonstrates the specificity of this thiol-disulfide oxidoreductase and enables us to outline a structural mechanism of regulating the usage of electrons in a thiol-disulfide oxidoreductase system.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Grupo Citocromo c/metabolismo , Proteína Disulfuro Reductasa (Glutatión)/química , Proteínas Bacterianas/metabolismo , Grupo Citocromo c/química , Transporte de Electrón , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Conformación Proteica , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Soluciones , Especificidad por Sustrato , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxin-like protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed.
Asunto(s)
Proteínas de Drosophila/química , Oxidación-Reducción , Selenoproteínas/química , Tiorredoxinas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Supervivencia Celular , Drosophila melanogaster , Escherichia coli/metabolismo , Proteínas de Insectos/química , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Células 3T3 NIH , Estrés Oxidativo , Conformación Proteica , Interferencia de ARN , Proteínas Recombinantes/química , Selenio/farmacología , Selenocisteína/química , Compuestos de SulfhidriloRESUMEN
Ubiquitin-conjugating enzymes (E2s) collaborate with the ubiquitin-activating enzyme (E1) and ubiquitin ligases (E3s) to attach ubiquitin to target proteins. RING-containing E3s simultaneously bind to E2s and substrates, bringing them into close proximity and thus facilitating ubiquitination. We show herein that, although the E3-binding site on the human E2 UbcH5b is distant from its active site, two RING-type minimal E3 modules lacking substrate-binding functions greatly stimulate the rate of ubiquitin release from the UbcH5b-ubiquitin thioester. Using statistical coupling analysis and mutagenesis, we identify and characterize clusters of coevolving and functionally linked residues within UbcH5b that span its E3-binding and active sites. Several UbcH5b mutants are defective in their stimulation by E3s despite their abilities to bind to these E3s, to form ubiquitin thioesters, and to release ubiquitin at a basal rate. One such mutation, I37A, is distant from both the active site and the E3-binding site of UbcH5b. Our studies reveal structural determinants for communication between distal functional sites of E2s and suggest that RING-type E3s activate E2s allosterically.
Asunto(s)
Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/química , Sitio Alostérico , Sitios de Unión , Análisis por Conglomerados , Ciclina B/química , Ciclina B1 , Cisteína/química , Escherichia coli/metabolismo , Ésteres/química , Humanos , Cinética , Ligasas/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis , Mutación , Péptidos/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Factores de Tiempo , Ubiquitina/química , Enzimas Activadoras de Ubiquitina/química , Complejos de Ubiquitina-Proteína Ligasa/químicaRESUMEN
The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity.