RESUMEN
Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.
RESUMEN
Molecular mechanisms underlying the thermal response of cells remain elusive. On the basis of the recent result that the short-time diffusive dynamics of the Escherichia coli proteome is an excellent indicator of temperature-dependent bacterial metabolism and death, we used neutron scattering (NS) spectroscopy and molecular dynamics (MD) simulations to investigate the sub-nanosecond proteome mobility in psychro-, meso-, and hyperthermophilic bacteria over a wide temperature range. The magnitude of thermal fluctuations, measured by atomic mean square displacements, is similar among all studied bacteria at their respective thermal cell death. Global roto-translational motions turn out to be the main factor distinguishing the bacterial dynamical properties. We ascribe this behavior to the difference in the average proteome net charge, which becomes less negative for increasing bacterial thermal stability. We propose that the chemical-physical properties of the cytoplasm and the global dynamics of the resulting proteome are fine-tuned by evolution to uphold optimal thermal stability conditions.