RESUMEN

Human hemoglobin (Hb) Coimbra (ßAsp99Glu) is one of the seven ßAsp99 Hb variants described to date. All ßAsp99 substitutions result in increased affinity for O2 and decreased heme-heme cooperativity and their carriers are clinically characterized by erythrocytocis, caused by tissue hypoxia. Since ßAsp99 plays an important role in the allosteric α1ß2 interface and the mutation in Hb Coimbra only represents the insertion of a CH2 group in this interface, the present study of Hb Coimbra is important for a better understanding of the global impact of small modifications in this allosteric interface. We carried out functional, kinetic and dynamic characterization of this hemoglobin, focusing on the interpretation of these results in the context of a growth of the position 99 side chain length in the α1ß2 interface. Oxygen affinity was evaluated by measuring p50 values in distinct pHs (Bohr effect), and the heme-heme cooperativity was analyzed by determining the Hill coefficient (n), in addition to the effect of the allosteric effectors inositol hexaphosphate (IHP) and 2,3-bisphosphoglyceric acid (2,3-BPG). Computer simulations revealed a stabilization of the R state in the Coimbra variant with respect to the wild type, and consistently, the T-to-R quaternary transition was observed on the nanosecond time scale of classical molecular dynamics simulations.

Asunto(s)

Hemoglobinas Anormales/química , Hemoglobinas Anormales/metabolismo , 2,3-Difosfoglicerato/farmacología , Regulación Alostérica , Hemo/metabolismo , Hemoglobinas Anormales/genética , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Oxígeno/metabolismo , Ácido Fítico/farmacología , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína

RESUMEN

Thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily of ligand-activated transcription factors involved in cell differentiation, growth, and homeostasis. Although X-ray structures of many nuclear receptor ligand-binding domains (LBDs) reveal that the ligand binds within the hydrophobic core of the ligand-binding pocket, a few studies suggest the possibility of ligands binding to other sites. Here, we report a new x-ray crystallographic structure of TR-LBD that shows a second binding site for T3 and T4 located between H9, H10, and H11 of the TRα LBD surface. Statistical multiple sequence analysis, site-directed mutagenesis, and cell transactivation assays indicate that residues of the second binding site could be important for the TR function. We also conducted molecular dynamics simulations to investigate ligand mobility and ligand-protein interaction for T3 and T4 bound to this new TR surface-binding site. Extensive molecular dynamics simulations designed to compute ligand-protein dissociation constant indicate that the binding affinities to this surface site are of the order of the plasma and intracellular concentrations of the thyroid hormones, suggesting that ligands may bind to this new binding site under physiological conditions. Therefore, the second binding site could be useful as a new target site for drug design and could modulate selectively TR functions.

Asunto(s)

Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/metabolismo , Aminoácidos/metabolismo , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Humanos , Ligandos , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Receptores de Hormona Tiroidea/genética , Relación Estructura-Actividad , Activación Transcripcional

RESUMEN

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH3. We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes.

Asunto(s)

Medición de Intercambio de Deuterio , Receptores de Hormona Tiroidea/agonistas , Receptores de Hormona Tiroidea/antagonistas & inhibidores , Secuencia de Aminoácidos , Amoníaco/farmacología , Apoproteínas/química , Apoproteínas/metabolismo , Deuterio/metabolismo , Humanos , Ligandos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación/genética , Péptidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Hormona Tiroidea/química , Alineación de Secuencia , Solventes , Triyodotironina/farmacología

RESUMEN

We report classical and tight-binding molecular dynamics simulations of the C(60) fullerene and cubane molecular crystal in order to investigate the intermolecular dynamics and polymerization processes. Our results show that, for 200 and 400 K, cubane molecules remain basically fixed, presenting only thermal vibrations, while C(60) fullerenes show rotational motions. Fullerenes perform "free" rotational motions at short times (approximately < 1 ps), small amplitude hindered rotational motions (librations) at intermediate times, and rotational diffusive dynamics at long times (approximately > 10 ps). The mechanisms underlying these dynamics are presented. Random copolymerizations among cubanes and fullerenes were observed when temperature is increased, leading to the formation of a disordered structure. Changes in the radial distribution function and electronic density of states indicate the coexistence of amorphous and crystalline phases. The different conformational phases that cubanes and fullerenes undergo during the copolymerization process are discussed.