RESUMEN
Microtubule doublets (MTDs), consisting of an incomplete B-microtubule at the surface of a complete A-microtubule, provide a structural scaffold mediating intraflagellar transport and ciliary beating. Despite the fundamental role of MTDs, the molecular mechanism governing their formation is unknown. We used a cell-free assay to demonstrate a crucial inhibitory role of the carboxyl-terminal (C-terminal) tail of tubulin in MTD assembly. Removal of the C-terminal tail of an assembled A-microtubule allowed for the nucleation of a B-microtubule on its surface. C-terminal tails of only one A-microtubule protofilament inhibited this side-to-surface tubulin interaction, which would be overcome in vivo with binding protein partners. The dynamics of B-microtubule nucleation and its distinctive isotropic elongation was elucidated by using live imaging. Thus, inherent interaction properties of tubulin provide a structural basis driving flagellar MTD assembly.
Asunto(s)
Cilios/ultraestructura , Microtúbulos/ultraestructura , Tubulina (Proteína)/química , Animales , Bovinos , Cilios/química , Simulación por Computador , Microscopía por Crioelectrón , Técnica del Anticuerpo Fluorescente , Microtúbulos/química , Modelos Moleculares , Unión Proteica , Subtilisina , Porcinos , Tetrahymena thermophilaRESUMEN
Biophysical techniques developed during the last three decades have provided an increasingly detailed description of the internal processes associated with ligand capture and release by haem proteins. Myoglobin has long been the paradigm for these studies. More recently, cytochrome P450cam (the camphor-metabolizing cytochrome P450 from Pseudomonas putida) has also received considerable interest. In spite of sharing the same prosthetic group, the Fe(II)-haem, these proteins are structurally unrelated and they perform different functions. Recent works show that both proteins exhibit a common feature: a series of permanent or fluctuating, mostly hydrophobic, cavities of the protein matrix are providing transient docking sites as well as migration, escape and possibly entry pathways for the ligand. Remarkably, these systems of cavities connect the distal and the proximal regions of the haem, a disposition that may contribute to ligand capture enhancement.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Hemoproteínas/metabolismo , Proteínas Bacterianas/metabolismo , Monóxido de Carbono/metabolismo , Sistema Enzimático del Citocromo P-450/química , Ligandos , Mioglobina/química , Mioglobina/metabolismo , Conformación ProteicaRESUMEN
The normal mode analysis of human hemoglobin showed the presence in the deoxy T-state of one main preferential direction that brings the structure close to the R-state, with a low-energy variation, while in the oxy R-state there are several modes that point towards the T-state, but with higher energy variations and less contribution to the transition. The displacement along a combination of normal modes, followed by energy minimization, starting from the R-state, did not allow one to obtain a structure significantly different from that of R, showing that the fully oxygenated hemoglobin is trapped in a deep and narrow potential energy minimum. On the contrary, starting from the deoxy T-state, the displacement along a combination of normal modes, followed by energy minimization, yielded an intermediate structure, that we designate Tmin(d1), which is closer to R; the normal modes of Tmin(d1) indicated that the potential energy minimum in the vicinity of this structure is as narrow as that of R but less deep. The procedure of displacement along the modes, followed by energy minimization, was applied to Tmin(d1), yielding Tmin(d2); then the procedure was repeated, yielding the intermediate structures Tmin(d3) and Tmin(d4). The structures Tmin(d2), Tmin(d3) and Tmin(d4) are not significantly different from each other, indicating that they are trapped in a narrow, deep energy minimum. This procedure revealed the existence of at least two intermediate sets of structures between T and R: the first one, Tmin(d1), is different from the T and R structures, while the second set, Tmin(d2), Tmin(d3) and Tmin(d4), is quaternary R-like and tertiary T-like, where the contacts at the interfaces alpha1 beta1 and alpha1 beta2 are R-like, and the alpha and beta heme environments are still T-like.
Asunto(s)
Hemoglobina A/química , Conformación Proteica , Hemo/química , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Aspartate transcarbamylase (ATCase) is an important control enzyme in the pyrimidine biosynthetic pathway in Escherichia coli. It is a classic example of an allosteric protein and has been extensively studied biochemically, kinetically and structurally. As yet, however, a detailed model for the cooperative transition between the tensed (T) and relaxed (R) forms of the protein does not exist. In this work we have calculated the low frequency normal modes of the CTP-ligated T-state of ATCase with the aim of identifying some of the motions that could be important in initiating the transition. The calculated modes, of frequencies lower than 5 per cm, produce root-mean-square coordinate deviations for the atoms which are a substantial fraction of those derived from the crystallographic B-factors. Some of the modes result in displacements which change the quaternary structure of the protein (in particular the elongation of the protein and the relative rotation of the subunits) in such a way that the R-state structure is approached. The implication of these mode motions for the overall T-->R transition process is discussed.
Asunto(s)
Aspartato Carbamoiltransferasa/química , Escherichia coli/enzimología , Sitio Alostérico , Sitios de Unión , Gráficos por Computador , Cristalografía por Rayos X , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
The method of diagonalization in a mixed basis (DIMB) that was published previously (Mouawad.), L. and Perahia D., Biopolymers 33, 599, 1993), allows the computation of the low-frequency vibrational modes for large macromolecules. Improvements to this method are presented here, namely the single and double truncation window techniques. The best convergence rate is obtained with the double truncation windows, which couple most efficiently the parts of the macromolecule which are far in sequence but close in space. Both methods were applied to the T-state of hemoglobin, to compare their efficiency. The resulting modes are analyzed in order to study the pathways between T- and R-states of this protein. They show that the quaternary conformational are mainly due to one mode at 2 cm-1.
Asunto(s)
Hemoglobinas/química , Conformación Proteica , Modelos Químicos , Programas InformáticosRESUMEN
This paper presents the comparative comprehensive analysis of NMR structural parameters (NOEs, scalar coupling, chemical shifts) of toxin gamma, a cardiotoxin isolated from the venom of Naja nigricollis, and three chemical derivatives, i.e. the 2-nitrophenylsulphonyl (NPS)-Trp11, 3-nitro-Tyr22 and 3-nitro-Tyr51 derivatives. In previous work, the chemical modifications of single side chains have suggested that these aromatic residues, in association with several lysine residues, contributed to the cytotoxicity of toxin gamma. Analysis of these results based on the refined solution structure of the toxin has resulted in the proposal of a conserved phospholipid binding site through which cardiotoxins are likely to interact with the membrane of target cells. The present work shows that modifications of either the tryptophan residue or the tyrosine residues, which are within or near the proposed binding site, have no influence on the three-dimensional structure of the protein. On the other hand, the proton exchange study of the backbone amides indicates that the structural core of the protein is destabilized in the three derivatives. This corresponds to a decrease of the overall stability of the protein as indicated by the comparative solvent denaturation study of the unmodified toxin gamma and the Trp11 derivative. More specifically, the dynamics of the three-stranded beta sheet, a part of the structural core, are highly perturbed by the chemical modifications. This sheet was previously proposed as a part of the phospholipid binding site of cardiotoxins. The dynamical perturbation of this site appears to be correlated with the decrease in toxicity of the chemical derivatives.
Asunto(s)
Proteínas Cardiotóxicas de Elápidos/química , Venenos Elapídicos/química , Nitrobencenos/química , Estructura Secundaria de Proteína , Animales , Unión Competitiva , Elapidae , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Protones , Solventes , Relación Estructura-Actividad , Tripsina/química , Tirosina/químicaRESUMEN
Examination of the literature has revealed that regarding the amino acid sequences, cardiotoxins constitute a family of homogeneous compounds. In contrast, cardiotoxins appear heterogeneous as far as their biological and spectroscopic properties are concerned. As a result, comparison between these molecules with a view to establishing structure-activity correlations is complicated. We have therefore reviewed recent works aiming at identifying the functional site of a defined cardiotoxin, ie toxin gamma from the venom of the spitting cobra Naja nigricollis. The biological and structural properties of toxin gamma are first described. In particular, a model depicting the 3-dimensional structure of the toxin studied by NMR spectroscopy is proposed. The toxin polypeptide chain is folded into 3 adjacent loops rich in beta-sheet structure connected to a small globular core containing the 4 disulfide bonds. A number of derivatives chemically modified at a single aromatic or amino group have been prepared. The structure of each derivative was probed by emission fluorescence, circular dichroism and NMR spectroscopy. Also tested was the ability of the derivatives to kill mice, depolarize excitable cell membranes and lyse epithelial cells. Modification of some residues in the first loop, in particular Lys-12 and at the base of the second loop substantially affected biological properties, with no sign of concomitant structural modifications other than local changes. Modifications in other regions much less affected the biological properties of the toxin. A plausible functional site for toxin gamma involving loop I and the base of loop II is presented. It is stressed that the functional site of other cardiotoxins may be different.
Asunto(s)
Proteínas Cardiotóxicas de Elápidos/química , Venenos Elapídicos/química , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Espectrofotometría , Relación Estructura-ActividadRESUMEN
Flexibility and folding of phosphoglycerate kinase, a two-domain monomeric enzyme, have been studied using a wide variety of methods including theoretical approaches. Mutants of yeast phosphoglycerate kinase have been prepared in order to introduce cysteinyl residues as local probes throughout the molecule without perturbating significantly the structural or the functional properties of the enzyme. The apparent reactivity of a unique cysteine in each mutant has been used to study the flexibility of PGK. The regions of larger mobility have been found around residue 183 on segment beta F in the N-domain and residue 376 on helix XII in the C-domain. These regions are also parts of the molecule which unfold first. Ligand binding induces conformational motions in the molecule, especially in the regions located in the cleft. Moreover, the results obtained by introducing a fluorescent probe covalently linked to a cysteine are in agreement with the helix scissor motion of helices 7 and 14 assumed by Blake to direct the hinge bending motion of the domains during the catalytic cycle. The folding process of both horse muscle and yeast phosphoglycerate kinases involves intermediates. These intermediates are more stable in the horse muscle than in the yeast enzyme. In both enzymes, domains behave as structural modules capable of folding and stabilizing independently, but in the horse muscle enzyme the C-domain is more stable and refolds prior to the N-domain, contrary to that which has been observed in the yeast enzyme. A direct demonstration of the independence of domains in yeast phosphoglycerate kinase has been provided following the obtention of separated domains by site-directed mutagenesis. These domains have a native-like structure and refold spontaneously after denaturation by guanidine hydrochloride.
Asunto(s)
Fosfoglicerato Quinasa/química , Animales , Sitios de Unión , Caballos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfoglicerato Quinasa/genética , Fosfoglicerato Quinasa/metabolismo , Conformación Proteica , Desnaturalización Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
Several mutants of yeast phosphoglycerate kinase, each containing only one internal cysteine residue, were constructed from a single mutant devoid of cysteine. These cysteines were introduced as local conformational probes in selected buried positions. The enzyme activity, conformational characteristics and stability indicated that the mutations introduced only small perturbations in the molecule. The folding-unfolding process mediated by guanidine hydrochloride under equilibrium conditions was studied by following the variations in ellipticity and the reactivity of the cysteine residue towards 5,5'-dithiobis(nitrobenzoate). The process was found to be reversible except for mutant C97A, V49C, suggesting that this region located in helix I might be crucial in determining an intermediate on the folding pathway. The transitions obtained by the two signals did not coincide, indicating that the local structures, in several parts inside the molecule, are more sensitive to the denaturant than the overall conformation.
Asunto(s)
Cisteína , Fosfoglicerato Quinasa/metabolismo , Saccharomyces cerevisiae/enzimología , Catálisis , Dicroismo Circular , Estabilidad de Medicamentos , Guanidina , Guanidinas/farmacología , Cinética , Estructura Molecular , Mutación , Fosfoglicerato Quinasa/genética , Conformación Proteica/efectos de los fármacos , Saccharomyces cerevisiae/genética , Compuestos de Sulfhidrilo , TermodinámicaRESUMEN
Horse muscle phosphoglycerate kinase (PGK) is a monomer folded into two widely distant domains. In the glycolytic pathway, this enzyme catalyzes the first reaction that produces ATP. It was suggested, by analogy with yeast hexokinase, that a hinge-bending motion may be induced by the binding of specific substrates to the protein. To analyze such a motion, or any structural changes induced by ligand binding, fluorescence anisotropy decay of tryptophan residues in free and liganded PGK was studied. At 293 K, for the free protein and the binary complex with 3-phosphoglycerate, a single correlation time of 26 ns was observed, corresponding to the rotation of the overall protein, whereas upon addition of MgADP, this correlation time decreased to 10 ns. Such a decrease cannot be merely due to a change of the protein's shape and volume. To explain this, it was suggested that the fluorescence anisotropy decay of the PGK-MgADP complex corresponded to the rotation of the only buried tryptophan (Trp 335). The rotational paths of this tryptophan, in the presence and absence of the nucleotide, were established by potential energy minimization calculations. The results indicated that MgADP induces a displacement of helix alpha-13 that decreases the rotational energy barrier of Trp 335 from 16 kcal/mol in the free protein to 8 kcal/mol in the complex.
Asunto(s)
Fosfoglicerato Quinasa/química , Adenosina Difosfato/metabolismo , Animales , Birrefringencia , Polarización de Fluorescencia , Ácidos Glicéricos/metabolismo , Glucólisis , Caballos , Isomerismo , Cinética , Ligandos , Sustancias Macromoleculares , Fosfoglicerato Quinasa/metabolismo , Conformación Proteica , Espectrofotometría Ultravioleta , Temperatura , Triptófano/metabolismoRESUMEN
Horse muscle phosphoglycerate kinase, like other mammalian phosphoglycerate kinases, contains seven cysteine residues of which two react rapidly with 5,5'-dithio-bis(2-nitrobenzoate) (Nbs2) following second-order kinetics (k = 640 M-1.s-1). Selective cyanylation of the fast-reacting cysteines, followed by chemical cleavage and subsequent sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis of the resulting polypeptides, suggested that these cysteines are at positions 378 and 379. Cysteine residues were introduced into yeast phosphoglycerate kinase by site-directed mutagenesis. Mutant enzymes, each containing only one cysteine residue at position 364, 376, or 377, were constructed from a mutant devoid of cysteine (Cys97----Ala). In the last two mutants, the cysteines were at positions corresponding to Cys378 and Cys379, respectively, in the horse muscle enzyme. The chemical reactivity of the cysteine groups in these latter two yeast mutant enzymes was similar to that of the fast-reacting cysteines in the horse muscle enzyme. Furthermore, they were similarly modified upon substrate binding. All these data demonstrate unambiguously that the fast-reacting cysteines in the horse muscle enzyme are Cys378 and Cys379.