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1.
J Am Chem Soc ; 141(6): 2367-2375, 2019 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-30657661

RESUMEN

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster between +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other. To our knowledge, this is the first example of a protein bound [Fe-S] cluster with three different amino acid side chains as ligands, and of Glu acting as ligand to a [2Fe-2S] cluster. Analyses of RsrR structures revealed a conformational change, centered on Trp9, which results in a significant shift in the DNA-binding helix-turn-helix region.


Asunto(s)
Proteínas Bacterianas/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN/metabolismo , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Factores de Transcripción/metabolismo
2.
J Am Chem Soc ; 138(36): 11802-9, 2016 09 14.
Artículo en Inglés | MEDLINE | ID: mdl-27545412

RESUMEN

The enzyme NadA catalyzes the synthesis of quinolinic acid (QA), the precursor of the universal nicotinamide adenine dinucleotide (NAD) cofactor. Here, we report the crystal structures of complexes between the Thermotoga maritima (Tm) NadA K219R/Y107F variant and (i) the first intermediate (W) resulting from the condensation of dihydroxyacetone phosphate (DHAP) with iminoaspartate and (ii) the DHAP analogue and triose-phosphate isomerase inhibitor phosphoglycolohydroxamate (PGH). In addition, using the TmNadA K219R/Y21F variant, we have reacted substrates and obtained a crystalline complex between this protein and the QA product. We also show that citrate can bind to both TmNadA K219R and its Y21F variant. The W structure indicates that condensation causes dephosphorylation. We propose that catalysis by the K219R/Y107F variant is arrested at the W intermediate because the mutated protein is unable to catalyze its aldo-keto isomerization and/or cyclization that ultimately lead to QA formation. Intriguingly, PGH binds to NadA with its phosphate group at the site where the carboxylate groups of W also bind. Our results shed significant light on the mechanism of the reaction catalyzed by NadA.


Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Ácido Quinolínico/metabolismo , Transferasas Alquil y Aril/genética , Cristalografía por Rayos X , Dihidroxiacetona Fosfato/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Conformación Proteica , Thermotoga maritima/enzimología
3.
Proc Natl Acad Sci U S A ; 109(14): 5305-10, 2012 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-22431599

RESUMEN

The crystal structure of the membrane-bound O(2)-tolerant [NiFe]-hydrogenase 1 from Escherichia coli (EcHyd-1) has been solved in three different states: as-isolated, H(2)-reduced, and chemically oxidized. As very recently reported for similar enzymes from Ralstonia eutropha and Hydrogenovibrio marinus, two supernumerary Cys residues coordinate the proximal [FeS] cluster in EcHyd-1, which lacks one of the inorganic sulfide ligands. We find that the as-isolated, aerobically purified species contains a mixture of at least two conformations for one of the cluster iron ions and Glu76. In one of them, Glu76 and the iron occupy positions that are similar to those found in O(2)-sensitive [NiFe]-hydrogenases. In the other conformation, this iron binds, besides three sulfur ligands, the amide N from Cys20 and one Oε of Glu76. Our calculations show that oxidation of this unique iron generates the high-potential form of the proximal cluster. The structural rearrangement caused by oxidation is confirmed by our H(2)-reduced and oxidized EcHyd-1 structures. Thus, thanks to the peculiar coordination of the unique iron, the proximal cluster can contribute two successive electrons to secure complete reduction of O(2) to H(2)O at the active site. The two observed conformations of Glu76 are consistent with this residue playing the role of a base to deprotonate the amide moiety of Cys20 upon iron binding and transfer the resulting proton away, thus allowing the second oxidation to be electroneutral. The comparison of our structures also shows the existence of a dynamic chain of water molecules, resulting from O(2) reduction, located near the active site.


Asunto(s)
Cristalografía por Rayos X/métodos , Escherichia coli/enzimología , Hidrogenasas/metabolismo , Oxígeno/metabolismo , Sitios de Unión , Hidrogenasas/química , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica
4.
J Am Chem Soc ; 136(14): 5253-6, 2014 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-24650327

RESUMEN

Quinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide (NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one substrate analog, providing partial mechanistic information. Here, we report on the holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases. Our mutagenesis data show that all of these residues are essential for activity and strongly suggest that Tyr21 deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA displays a dehydration mechanism significantly different from the one found in archetypical dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The X-ray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of NAD biosynthesis.


Asunto(s)
Hidrolasas/química , Complejos Multienzimáticos/química , Tirosina/química , Cristalografía por Rayos X , Deshidratación , Hidrolasas/metabolismo , Modelos Moleculares , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Tirosina/metabolismo
5.
Sci Adv ; 9(12): eade9674, 2023 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-36961893

RESUMEN

Most bacteriophages present a tail allowing host recognition, cell wall perforation, and viral DNA channeling from the capsid to the infected bacterium cytoplasm. The majority of tailed phages bear a long flexible tail (Siphoviridae) at the tip of which receptor binding proteins (RBPs) specifically interact with their host, triggering infection. In siphophage T5, the unique RBP is located at the extremity of a central fiber. We present the structures of T5 tail tip, determined by cryo-electron microscopy before and after interaction with its E. coli receptor, FhuA, reconstituted into nanodisc. These structures bring out the important conformational changes undergone by T5 tail tip upon infection, which include bending of T5 central fiber on the side of the tail tip, tail anchoring to the membrane, tail tube opening, and formation of a transmembrane channel. The data allow to detail the first steps of an otherwise undescribed infection mechanism.


Asunto(s)
Bacteriófagos , Siphoviridae , Bacteriófagos/genética , Escherichia coli/metabolismo , Microscopía por Crioelectrón , Siphoviridae/química , Pared Celular
6.
Photochem Photobiol Sci ; 9(2): 254-62, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20126803

RESUMEN

We have studied the photoswitching behaviour of a number of photochromic fluorescent proteins at cryo-temperature. Spectroscopic investigations at the ensemble level showed that EYFP, Dronpa and IrisFP all exhibit reversible photoswitching at 100 K, albeit with a low quantum yield. The photophysics of the process were studied in more details in the case of EYFP. The data suggest that photoinduced protonation of the chromophore is responsible for off-switching at cryo-temperature, and thus is possible in the absence of significant conformational freedom. This finding is consistent with the hypothesis that chromophore protonation may precede large amplitude conformational changes such as cis-trans isomerisation during off-photoswitching at room temperature. However, our data suggest that low-barrier photoinduced protonation pathways may in fact compete with room-temperature off-switching reactions in photochromic fluorescent proteins. The occurrence of reversible photoswitching at low-temperature is of interest to envisage cryo-nanoscopy experiments using genetically encoded fluorophores.


Asunto(s)
Proteínas Luminiscentes/química , Protones , Proteínas Bacterianas/química , Frío , Isomerismo , Teoría Cuántica , Espectrofotometría Ultravioleta
7.
Biophys J ; 96(5): 1902-10, 2009 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-19254549

RESUMEN

Solvent fluctuations play a key role in controlling protein motions and biological function. Here, we have studied how individual steps of the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (POR) couple with solvent dynamics. To simultaneously monitor the catalytic cycle of the enzyme and the dynamical behavior of the solvent, we designed temperature-dependent UV-visible microspectrophotometry experiments, using flash-cooled nanodroplets of POR to which an exogenous soluble fluorophore was added. The formation and decay of the first two intermediates in the POR-catalyzed reaction were measured, together with the solvent glass transition and the buildup of crystalline ice at cryogenic temperatures. We find that formation of the first intermediate occurs below the glass transition temperature (T(g)), and is not affected by changes in solvent dynamics induced by modifying the glycerol content. In contrast, formation of the second intermediate occurs above T(g) and is influenced by changes in glycerol concentration in a manner remarkably similar to the buildup of crystalline ice. These results suggest that internal, nonslaved protein motions drive the first step of the POR-catalyzed reaction whereas solvent-slaved motions control the second step. We propose that the concept of solvent slaving applies to complex enzymes such as POR.


Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Solventes/química , Algoritmos , Proteínas Bacterianas/química , Cianobacterias/enzimología , Polarización de Fluorescencia , Cinética , Luz , Microespectrofotometría/métodos , Modelos Químicos , Conformación Proteica , Espectrofotometría Ultravioleta/métodos , Temperatura , Viscosidad
8.
Biochemistry ; 48(33): 7916-26, 2009 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-19650626

RESUMEN

Ni-dependent acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) constitute the central enzyme complex of the Wood-Ljungdahl pathway of acetyl-CoA formation. The crystal structure of a recombinant bacterial ACS lacking the N-terminal domain that interacts with CODH shows a large reorganization of the remaining two globular domains, producing a narrow cleft of suitable size, shape, and nature to bind CoA. Sequence comparisons with homologous archaeal enzymes that naturally lack the N-terminal domain show that many amino acids lining this cleft are conserved. Besides the typical [4Fe-4S] center, the A-cluster contains only one proximal metal ion that, according to anomalous scattering data, is most likely Cu or Zn. Incorporation of a functional Ni(2)Fe(4)S(4) A-cluster would require only minor structural rearrangements. Using available structures, a plausible model of the interaction between CODH and the smaller ACS in archaeal multienzyme complexes is presented, along with a discussion of evolutionary relationships of the archaeal and bacterial enzymes.


Asunto(s)
Acetato CoA Ligasa/química , Bacterias Grampositivas/enzimología , Eliminación de Secuencia , Acetato CoA Ligasa/genética , Acetato CoA Ligasa/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Bacterianas/química , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Bacterias Grampositivas/genética , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína/genética
9.
J Am Chem Soc ; 131(50): 18063-5, 2009 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-19950947

RESUMEN

We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV-vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of pi conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles' heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants.


Asunto(s)
Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/efectos de la radiación , Fotoblanqueo , Cristalografía por Rayos X , Transporte de Electrón , Modelos Químicos , Conformación Proteica , Espectrometría Raman , Rayos X
10.
Chem Commun (Camb) ; 55(26): 3725-3728, 2019 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-30855610

RESUMEN

Quinolinate synthase (NadA) is a [4Fe-4S] cluster-containing enzyme involved in the formation of quinolinic acid, the precursor of the essential NAD coenzyme. Here, we report the synthesis and activity of derivatives of the first inhibitor of NadA. Using multidisciplinary approaches we have investigated their action mechanism and discovered additional specific inhibitors of this enzyme.

11.
Structure ; 14(1): 129-39, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16407072

RESUMEN

Iron regulatory proteins (IRPs) control the translation of proteins involved in iron uptake, storage and utilization by binding to specific noncoding sequences of the corresponding mRNAs known as iron-responsive elements (IREs). This strong interaction assures proper iron homeostasis in animal cells under iron shortage. Conversely, under iron-replete conditions, IRP1 binds a [4Fe-4S] cluster and functions as cytosolic aconitase. Regulation of the balance between the two IRP1 activities is complex, and it does not depend only on iron availability. Here, we report the crystal structure of human IRP1 in its aconitase form. Comparison with known structures of homologous enzymes reveals well-conserved folds and active site environments with significantly different surface shapes and charge distributions. The specific features of human IRP1 allow us to propose a tentative model of an IRP1-IRE complex that agrees with a range of previously obtained data.


Asunto(s)
Aconitato Hidratasa/química , Citosol/enzimología , Proteína 1 Reguladora de Hierro/química , Aconitato Hidratasa/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Bovinos , Cristalización , Cristalografía por Rayos X , Dimerización , Humanos , Proteína 1 Reguladora de Hierro/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia
12.
ACS Chem Biol ; 13(5): 1209-1217, 2018 05 18.
Artículo en Inglés | MEDLINE | ID: mdl-29641168

RESUMEN

NadA is a multifunctional enzyme that condenses dihydroxyacetone phosphate (DHAP) with iminoaspartate (IA) to generate quinolinic acid (QA), the universal precursor of the nicotinamide adenine dinucleotide (NAD(P)) cofactor. Using X-ray crystallography, we have (i) characterized two of the reaction intermediates of QA synthesis using a "pH-shift" approach and a slowly reacting Thermotoga maritima NadA variant and (ii) observed the QA product, resulting from the degradation of an intermediate analogue, bound close to the entrance of a long tunnel leading to the solvent medium. We have also used molecular docking to propose a condensation mechanism between DHAP and IA based on two previously published Pyrococcus horikoshi NadA structures. The combination of reported data and our new results provide a structure-based complete catalytic sequence of QA synthesis by NadA.


Asunto(s)
Complejos Multienzimáticos/química , Ácido Quinolínico/metabolismo , Thermotoga maritima/enzimología , Cristalografía por Rayos X , Simulación del Acoplamiento Molecular , Complejos Multienzimáticos/metabolismo , NAD/metabolismo , Conformación Proteica
13.
Nat Commun ; 8: 15052, 2017 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-28425466

RESUMEN

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe-4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe-4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Hierro-Azufre/metabolismo , Óxido Nítrico/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Cristalografía por Rayos X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Modelos Moleculares , Conformación Proteica , Homología de Secuencia de Aminoácido , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética
14.
Sci Adv ; 1(11): e1501086, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26665177

RESUMEN

The structure of the dimeric holo-fumarate and nitrate reduction regulator (FNR) from Aliivibrio fischeri has been solved at 2.65 Å resolution. FNR globally controls the transition between anaerobic and aerobic respiration in facultative anaerobes through the assembly/degradation of its oxygen-sensitive [4Fe-4S] cluster. In the absence of O2, FNR forms a dimer and specifically binds to DNA, whereas in its presence, the cluster is degraded causing FNR monomerization and DNA dissociation. We have used our crystal structure and the information previously gathered from numerous FNR variants to propose that this process is governed by extremely fine-tuned interactions, mediated by two salt bridges near the amino-terminal cluster-binding domain and an "imperfect" coiled-coil dimer interface. [4Fe-4S] to [2Fe-2S] cluster degradation propagates a conformational signal that indirectly causes monomerization by disrupting the first of these interactions and unleashing the "unzipping" of the FNR dimer in the direction of the carboxyl-terminal DNA binding domain.

15.
Structure ; 21(1): 184-190, 2013 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-23260654

RESUMEN

We report the 3.3 Å resolution structure of dimeric membrane-bound O(2)-tolerant hydrogenase 1 from Escherichia coli in a 2:1 complex with its physiological partner, cytochrome b. From the short distance between distal [Fe(4)S(4)] clusters, we predict rapid transfer of H(2)-derived electrons between hydrogenase heterodimers. Thus, under low O(2) levels, a functional active site in one heterodimer can reductively reactivate its O(2)-exposed counterpart in the other. Hydrogenase 1 is maximally expressed during fermentation, when electron acceptors are scarce. These conditions are achieved in the lower part of the host's intestinal tract when E. coli is soon to be excreted and undergo an anaerobic-to-aerobic metabolic transition. The apparent paradox of having an O(2)-tolerant hydrogenase expressed under anoxia makes sense if the enzyme functions to keep intracellular O(2) levels low by reducing it to water, protecting O(2)-sensitive enzymes during the transition. Cytochrome b's main role may be anchoring the hydrogenase to the membrane.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Hidrogenasas/química , Dominio Catalítico , Cristalografía por Rayos X , Hemo/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Propiedades de Superficie
16.
J Immunol ; 180(4): 2329-38, 2008 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-18250442

RESUMEN

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute "eat me" signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance. HeLa cells were rendered apoptotic by UV-B treatment and a variety of cellular and molecular approaches were used to investigate the nature of the target(s) recognized by C1q. Using surface plasmon resonance, C1q binding was shown to occur at early stages of apoptosis and to involve recognition of a cell membrane component. C1q binding and phosphatidylserine (PS) exposure, as measured by annexin V labeling, proceeded concomitantly, and annexin V inhibited C1q binding in a dose-dependent manner. As shown by cosedimentation, surface plasmon resonance, and x-ray crystallographic analyses, C1q recognized PS specifically and avidly (K(D) = 3.7-7 x 10(-8) M), through multiple interactions between its globular domain and the phosphoserine group of PS. Confocal microscopy revealed that the majority of the C1q molecules were distributed in membrane patches where they colocalized with PS. In summary, PS is one of the C1q ligands on apoptotic cells, and C1q-PS interaction takes place at early stages of apoptosis, in newly organized membrane patches. Given its versatile recognition properties, these data suggest that C1q has the unique ability to sense different markers which collectively would provide strong eat me signals, thereby allowing efficient apoptotic cell removal.


Asunto(s)
Apoptosis/inmunología , Complemento C1q/metabolismo , Fosfatidilserinas/metabolismo , Complemento C1q/química , Cristalografía por Rayos X , Células HeLa , Humanos , Ligandos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie
17.
Nat Struct Biol ; 10(4): 271-9, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12627225

RESUMEN

The crystal structure of the tetrameric alpha2beta2 acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica has been solved at 1.9 A resolution. Surprisingly, the two alpha subunits display different (open and closed) conformations. Furthermore, X-ray data collected from crystals near the absorption edges of several metal ions indicate that the closed form contains one Zn and one Ni at its active site metal cluster (A-cluster) in the alpha subunit, whereas the open form has two Ni ions at the corresponding positions. Alternative metal contents at the active site have been observed in a recent structure of the same protein in which A-clusters contained one Cu and one Ni, and in reconstitution studies of a recombinant apo form of a related acetyl-CoA synthase. On the basis of our observations along with previously reported data, we postulate that only the A-clusters containing two Ni ions are catalytically active.


Asunto(s)
Acetato CoA Ligasa/química , Aldehído Oxidorreductasas/química , Complejos Multienzimáticos/química , Acetato CoA Ligasa/metabolismo , Acetilcoenzima A/biosíntesis , Aldehído Oxidorreductasas/metabolismo , Dominio Catalítico , Clostridium/enzimología , Cristalografía por Rayos X , Hierro/química , Modelos Biológicos , Modelos Moleculares , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Níquel/química , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Electricidad Estática , Azufre/química , Zinc/química
18.
Immunity ; 16(3): 345-54, 2002 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11911820

RESUMEN

The elongated complementary-determining region (CDR) 3beta found in the unliganded KB5-C20 TCR protrudes from the antigen binding site and prevents its docking onto the peptide/MHC (pMHC) surface according to a canonical diagonal orientation. We now present the crystal structure of a complex involving the KB5-C20 TCR and an octapeptide bound to the allogeneic H-2K(b) MHC class I molecule. This structure reveals how a tremendously large CDR3beta conformational change allows the KB5-C20 TCR to adapt to the rather constrained pMHC surface and achieve a diagonal docking mode. This extreme case of induced fit also shows that TCR plasticity is primarily restricted to CDR3 loops and does not propagate away from the antigen binding site.


Asunto(s)
Regiones Determinantes de Complementariedad/química , Antígenos de Histocompatibilidad Clase I/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Regiones Determinantes de Complementariedad/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Ratones , Modelos Moleculares , Conformación Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Relación Estructura-Actividad , Linfocitos T/química
19.
J Biol Chem ; 278(47): 46974-82, 2003 Nov 21.
Artículo en Inglés | MEDLINE | ID: mdl-12960167

RESUMEN

C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 A of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca2+ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.


Asunto(s)
Complemento C1q/química , Subunidades de Proteína/química , Proteína C-Reactiva/química , Proteína C-Reactiva/metabolismo , Calcio/química , Complemento C1q/metabolismo , Cristalización , Cristalografía por Rayos X , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Ligandos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismo
20.
Nat Immunol ; 4(3): 241-7, 2003 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-12563259

RESUMEN

T cell receptor (TCR) binding degeneracy lies at the heart of several physiological and pathological phenomena, yet its structural basis is poorly understood. We determined the crystal structure of a complex involving the BM3.3 TCR and an octapeptide (VSV8) bound to the H-2K(b) major histocompatibility complex molecule at a 2.7 A resolution, and compared it with the BM3.3 TCR bound to the H-2K(b) molecule loaded with a peptide that has no primary sequence identity with VSV8. Comparison of these structures showed that the BM3.3 TCR complementarity-determining region (CDR) 3alpha could undergo rearrangements to adapt to structurally different peptide residues. Therefore, CDR3 loop flexibility helps explain TCR binding cross-reactivity.


Asunto(s)
Regiones Determinantes de Complementariedad/química , Receptores de Antígenos de Linfocitos T/química , Linfocitos T/inmunología , Regiones Determinantes de Complementariedad/inmunología , Humanos , Ligandos , Unión Proteica/inmunología , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/inmunología , Relación Estructura-Actividad , Linfocitos T/química
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