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Institut de Biologie StructuraleGrenoble / France

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Insights into the movements of aromatic residues in a 0.5 MDa enzyme by solid-state NMR

Aromatic residues play key roles in many proteins, and are involved in protein-ligand and protein-protein interactions. They also can be found in the hydrophobic core of proteins, where they are crucial for protein stability. For these reasons, studying their dynamics can provide rich information on reaction mechanisms and folding. Although solution-state NMR has been used to study the motions of aromatic residues for decades, these studies have been limited to small proteins, due to inherent physical restrictions. A new approach developed by the IBS NMR group, in collaboration with Japanese and Austrian chemists, makes it possible to gain insights into the movements of aromatic residues even in very large proteins. The approach combines solid-state NMR and specific isotopic labelling of phenylalanines or tyrosines. The study by Gauto et al applied this method to the 468 kDa enzyme TET2 protein and demonstrated, among other things, the rotational kinetics of phenylalanines over a wide temperature range, down to -170°C. Temperature-dependent measurements reveal how the motions of the protein are activated when the temperature is increased, with an interesting appearance of motion at about -70 °C, often call the "glass-transition temperature". Furthermore, the approach allowed to infer "invisible" states with life times of microseconds, which, interestingly cluster around a pore located between subunits. This methodology also allows to obtain distance information between aromatic residues and their environment, which are valuable for determining the three-dimensional structures of proteins.

Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific 1H-13C Labeling and Fast Magic-Angle Spinning NMR. Gauto DF, Macek P, Barducci A, Fraga H, Hessel A, Terauchi T, Gajan D, Miyanoiri Y, Boisbouvier J, Lichtenecker R, Kainosho M, Schanda P. Journal of the American Chemical Society ; 141(28):11183-11195

New issue for the IBS Newsletter

Find the September 2019 issue (in french only).

Mechanism of allosteric activation of an enzyme by an inhibitor

The finding that an inhibitor activates an enzyme appears counterintuitive. How can an inhibitor, which binds to the active site, increase the enzyme’s activity ? In this study, researchers at IBS (NMR, MICA and PG groups), in collaboration with colleagues in Nancy and Zaragoza, revealed how the 300 kDa large protease ClpP undergoes this intriguing allosteric activation by an inhibitor as well as by substrates. A multi-technique approach, involving X-ray crystallography, solution- and solid-state NMR, molecular dynamics simulations and calorimetry, shows that substoichiometric binding of inhibitors to active sites shifts the equilibrium in this oligomeric protein to a more active extended state. The findings may provide new routes for the development of drugs of this potential antibiotic protein target.

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors. Felix J, Weinhäupl K, Chipot C, Dehez F, Hessel A, Gauto DF, Morlot C, Abian O, Gutsche I, Velazquez-Campoy A, Schanda P, Fraga H. Science Advances ; Vol. 5, no. 9, eaaw3818