Thermal activation of 'allosteric- like' large-scale motions in a eukaryotic Lactate Dehydrogenase - Archive ouverte HAL Access content directly
Journal Articles Scientific Reports Year : 2017

Thermal activation of 'allosteric- like' large-scale motions in a eukaryotic Lactate Dehydrogenase


Conformational changes occurring during the enzymatic turnover are essential for the regulation of protein functionality. Individuating the protein regions involved in these changes and the associated mechanical modes is still a challenge at both experimental and theoretical levels. We present here a detailed investigation of the thermal activation of the functional modes and conformational changes in a eukaryotic Lactate Dehydrogenase enzyme (LDH). Neutron Spin Echo spectroscopy and Molecular Dynamics simulations were used to uncover the characteristic length-and timescales of the LDH nanoscale motions in the apo state. The modes involving the catalytic loop and the mobile region around the binding site are activated at room temperature, and match the allosteric reorganisation of bacterial LDHs. In a temperature window of about 15 degrees, these modes render the protein flexible enough and capable of reorganising the active site toward reactive configurations. On the other hand an excess of thermal excitation leads to the distortion of the protein matrix with a possible anti-catalytic effect. Thus, the temperature activates eukaryotic LDHs via the same conformational changes observed in the allosteric bacterial LDHs. Our investigation provides an extended molecular picture of eukaryotic LDH's conformational landscape that enriches the static view based on crystallographic studies alone. Protein dynamics and functionality are intimately related. Nevertheless, the fine details of how the conforma-tional changes of proteins modulate and regulate their activity are still to be defined 1–4. It is now accepted that protein dynamics is characterized by a hierarchy of timescales, from picoseconds to microseconds, reflecting a rough manifold conformational landscape 5–7. There have been numerous studies on the relationship between this wide range of dynamical processes and protein functionality. These include substrate binding/unbinding kinetics 8 , catalysis 9,10 , and allosteric relaxation 11,12. To date, experimental techniques such as Nuclear Magnetic Resonance 7 , single molecule spectroscopy 10,13,14 , time-resolved X-ray crystallography 15 , and Neutron Scattering 16,17 represent the principal means of investigation of protein dynamics and function. Particularly, elastic, quasielastic, and inelastic incoherent NS have been exploited not only to study the sub-nanosecond timescale local functional dynamics of model proteins 18,19 and their solvent 20,21 , but also for in-vivo investigations of bacterial systems 22. On the other hand, Neutron Spin Echo spectroscopy (NSE) has been shown to be an invaluable tool to explore the dynamics of biomolecules on larger spatial scales, of the order of nanometer, for times up to hundreds of nanoseconds 23–27. NSE has been successfully applied to systems that exhibit long-range signaling modes via domain displacement, as in the case of the NHERF1 25 , Taq polymerase 23 , and Phosphoglycerate Kinase 26 , as well as to Alcohol Dehydrogenase, a more compact multimeric protein 24. Because the investigated modes involve length scales that match the size of the proteins , their dynamics overlap with the rigid body motions. Therefore, molecular modelling, e.g. Normal Mode (NM) analysis and Molecular Dynamics simulations (MD), are a necessary tool to disentangle the contribution of specific internal modes, and complement experiments.
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hal-01473622 , version 1 (22-02-2017)



Marina Katava, Marco Maccarini, Guillaume Villain, Alessandro Paciaroni, Michael Sztucki, et al.. Thermal activation of 'allosteric- like' large-scale motions in a eukaryotic Lactate Dehydrogenase. Scientific Reports, 2017, 7, pp.41092. ⟨10.1038/srep41092⟩. ⟨hal-01473622⟩
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