Estudio del Mecanismo de termodetección de DesK, una histidina quinasa termosensora de Bacillus subtilis
Fecha
2014-10-24
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Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas.
Resumen
El cambio en la temperatura ambiental afecta a las reacciones biológicas y las células deben adaptarse. Un ejemplo de ello es la regulación de la fluidez de la membrana. Las bacterias pueden remodelar la fluidez de la bicapa con precisión a través de la incorporación de una mayor proporción de ácidos grasos insaturados cuando la temperatura de crecimiento disminuye, esto altera el ordenamiento de la bicapa lipídica y optimiza la fluidez. Pero, ¿cómo pueden las células "sensar" la fluidez de la membrana? La histidina quinasa DesK de Bacillus subtilis es el ejemplo paradigmático de un sensor térmico capaz de remodelar la fluidez de la membrana cuando la temperatura cae por debajo de 30 ° C, proporcionando un sistema para investigar el mecanismo de adaptación térmica. Entonces, ¿cuál es el mecanismo por el cual DesK es capaz de detectar cambios de temperatura y cómo puede convertir esta señal física externa en una respuesta biológica interna?
Esta tesis estudia cómo la información térmica se integra, procesa y transduce para controlar la expresión génica y la intrigante posibilidad de que las deformaciones inducidas por la temperatura en la membrana celular actúen como reguladoras alostéricas. Como responsable del control por temperatura, encontramos una inestabilidad incorporada al sistema causada por un grupo de residuos hidrofílicos situados cerca del extremo N-terminal del primer segmento transmembrana, presumiblemente justo debajo de la interfase lípido/agua. Estos residuos enterrados en la fase lipídica a baja temperatura son capaces de bucear a la fase acuosa, estabilizando la posición del segmento transmembrana como una boya. En el extremo C-terminal de la parte transmembrana se detectó otro motivo sensible a la temperatura: el Cierre de Serina. El grosor de la membrana controla el estado de señalización del sensor al dictar el nivel de hidratación de este motivo hidrofílico metaestable. Nuestros resultados revelaron que DesK funciona como un dímero, en el que el motivo boya N-terminal actúa junto con un motivo hidrófilo C-terminal para causar una reorientación de las hélices en respuesta a cambios en el espesor de la membrana, dirigiendo la activación proteica. Sin embargo, la pregunta subyacente aún se mantiene: ¿Cómo puede la información detectada por la región transmembrana convertirse en un reordenamiento en el dominio catalítico citoplasmático para controlar la actividad de DesK? Aquí, identificamos una "región linker" implicada en la transmisión de la señal que conecta el transmembrana con el dominio citoplasmático. El linker adopta dos estados conformacionales en respuesta a los cambios de espesor de la membrana dependientes de la temperatura: (i) random coil, unido a las cabezas de los fosfolípidos, promoviendo el estado de la fosfatasa o (ii) no unido, formando una hélice continua que atraviesa la región de la membrana hasta el citoplasma, promoviendo así el estado quinasa. Nuestros resultados sostienen la idea de que el linker está dotado de una dualidad conformacional hélice/random coil que permite que se comporte como un interruptor de transmisión, con una discontinuidad en la hélice disminuyendo la relación de actividad quinasa/fosfatasa, como se requiere para modular la respuesta de DesK.
Environmental temperature variations affect most biological reactions, so cells must adapt accordingly. One such example is the fine-tuned regulation of membrane fluidity and its impact on the performance of a large array of cell physiological processes. Bacteria can remodel the fluidity of their membrane bilayer precisely via the incorporation of proportionally more unsaturated fatty acids as growth temperature decreases, this disrupts the order of the lipid bilayer and optimize fluidity. But, how do cells “sense” membrane fluidity? The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a polytopic membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. So, what is the mechanism by which DesK is able to sense temperature changes and how can it turn this external physical signal into biological inner response? This thesis focuses on how thermal information is integrated, processed, and transduced to control gene expression; how membrane-mediated structural changes of the cold sensor are accomplished; and the intriguing possibility that temperature-induced deformations of the cell membrane act as allosteric regulators of protein function. We found that temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane segment, presumably just below the lipid/water interface. These residues buried in the lipid phase at low temperature are able to snorkel to the aqueous phase, stabilizing the position of the transmembrane segment as a buoy. At the C-terminus of the transmembrane part another boundary-sensitive beacon was detected: a Serine Zipper motif. Membrane thickness controls the signaling state of the sensor by dictating the hydration level of this metastable hydrophilic spot. Our results revealed that DesK functions as a dimer, in which the N-terminal buoy motif acts together with the C-terminal hydrophilic motif to cause a reorientation of the helixes in response to changes in membrane thickness, leading to the activation of the protein. Nevertheless, the core question remained still poorly understood: How is the information sensed by the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain to control DesK activity? Here, we also identify a “linker region” that connects the TM sensor domain with the cytoplasmic catalytic domain involved in signal transmission. The linker adopts two conformational states in response to temperature-dependent membrane thickness changes: (i) random coiled and bound to the phospholipid head groups at the water-membrane interface, promoting the phosphatase state or (ii) unbound and forming a continuous helix spanning a region from the membrane to the cytoplasm, promoting the kinase state. Our results uphold the view that the linker is endowed with a helix/ random coil conformational duality that enables it to behave like a transmission switch, with helix disruption decreasing the kinase/ phosphatase activity ratio, as required to modulate the DesK output response. Because many sensors contain similar domains, the mechanism we describe here could prove a general theme.
Environmental temperature variations affect most biological reactions, so cells must adapt accordingly. One such example is the fine-tuned regulation of membrane fluidity and its impact on the performance of a large array of cell physiological processes. Bacteria can remodel the fluidity of their membrane bilayer precisely via the incorporation of proportionally more unsaturated fatty acids as growth temperature decreases, this disrupts the order of the lipid bilayer and optimize fluidity. But, how do cells “sense” membrane fluidity? The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a polytopic membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. So, what is the mechanism by which DesK is able to sense temperature changes and how can it turn this external physical signal into biological inner response? This thesis focuses on how thermal information is integrated, processed, and transduced to control gene expression; how membrane-mediated structural changes of the cold sensor are accomplished; and the intriguing possibility that temperature-induced deformations of the cell membrane act as allosteric regulators of protein function. We found that temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane segment, presumably just below the lipid/water interface. These residues buried in the lipid phase at low temperature are able to snorkel to the aqueous phase, stabilizing the position of the transmembrane segment as a buoy. At the C-terminus of the transmembrane part another boundary-sensitive beacon was detected: a Serine Zipper motif. Membrane thickness controls the signaling state of the sensor by dictating the hydration level of this metastable hydrophilic spot. Our results revealed that DesK functions as a dimer, in which the N-terminal buoy motif acts together with the C-terminal hydrophilic motif to cause a reorientation of the helixes in response to changes in membrane thickness, leading to the activation of the protein. Nevertheless, the core question remained still poorly understood: How is the information sensed by the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain to control DesK activity? Here, we also identify a “linker region” that connects the TM sensor domain with the cytoplasmic catalytic domain involved in signal transmission. The linker adopts two conformational states in response to temperature-dependent membrane thickness changes: (i) random coiled and bound to the phospholipid head groups at the water-membrane interface, promoting the phosphatase state or (ii) unbound and forming a continuous helix spanning a region from the membrane to the cytoplasm, promoting the kinase state. Our results uphold the view that the linker is endowed with a helix/ random coil conformational duality that enables it to behave like a transmission switch, with helix disruption decreasing the kinase/ phosphatase activity ratio, as required to modulate the DesK output response. Because many sensors contain similar domains, the mechanism we describe here could prove a general theme.
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Palabras clave
Mecanismo, Histidin kinasa, Termodetección