4 research outputs found
Interhelical H-Bonds Modulate the Activity of a Polytopic Transmembrane Kinase
DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a coexpression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.Fil: Almada, Juan Cruz. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa. Argentina.Fil: Bortolotti, Ana. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa. Argentina.Fil: Ruysschaert, Jean Marie. UniversitĂ© Libre de Bruxelles. Center for Structural Biology and Bioinformatics. Laboratory for the Structure and Function of Biological Membranes. Belgium.Fil: De Mendoza, Diego. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario (IBR-CONICET). Argentina.Fil: Inda, MarĂa Eugenia. Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science. Research Laboratory of Electronics. Cambridge.Fil: Inda, MarĂa Eugenia. Massachusetts Institute of Technology. MIT Synthetic Biology Center. Cambridge.Fil:Cybulski, Larisa EstefanĂa. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa. Argentina
A lipid-mediated conformational switch modulates the thermosensing activity of DesK.
The thermosensor DesK is a multipass transmembrane histidine-kinase that allows the bacterium Bacillus subtilis to adjust the levels of unsaturated fatty acids required to optimize membrane lipid fluidity. The cytoplasmic catalytic domain of DesK behaves like a kinase at low temperature and like a phosphatase at high temperature. Temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane (TM) segment. These residues are buried in the lipid phase at low temperature and partially "buoy" to the aqueous phase at higher temperature with the thinning of the membrane, promoting the required conformational change. Nevertheless, the core question remains 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 identify a "linker region" (KSRKERERLEEK) 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.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe
Driving the catalytic activity of a transmembrane thermosensor kinase
DesK is a Bacillus thermosensor kinase that is inactive at high temperatures but turns activated when the temperature drops below 25 °C. Surprisingly, the catalytic domain (DesKC) lacking the transmembrane region is more active at higher temperature, showing an inverted regulation regarding DesK. How does the transmembrane region control the catalytic domain, repressing activity at high temperatures, but allowing activation at lower temperatures? By designing a set of temperature minimized sensors that share the same catalytic cytoplasmic domain but differ in number and position of hydrogen-bond (H-bond) forming residues along the transmembrane helix, we are able to tune, invert or disconnect activity from the input signal. By favoring differential H-bond networks, the activation peak could be moved towards lower or higher temperatures. This principle may be involved in regulation of other sensors as environmental physicochemical changes or mutations that modify the transmembrane H-bond pattern can tilt the equilibrium favoring alternative conformations.Fil: Inda, MarĂa Eugenia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa; ArgentinaFil: Almada, Juan Cruz. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa; ArgentinaFil: Vázquez, Daniela BelĂ©n. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa; ArgentinaFil: Bortolotti, Ana. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa; ArgentinaFil: Fernandez, Ariel. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Saavedra 15. Instituto Argentino de Matemática Alberto CalderĂłn; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - BahĂa Blanca. Instituto de QuĂmica del Sur. Universidad Nacional del Sur. Departamento de QuĂmica. Instituto de QuĂmica del Sur; ArgentinaFil: Ruysschaert, Jean Marie. Campus de la Plaine; BĂ©lgicaFil: Cybulski, Larisa EstefanĂa. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Departamento de MicrobiologĂa; Argentin