Resolving conformational changes that mediate a two-step catalytic mechanism in a model enzyme

Abstract

Enzymes catalyze biochemical reactions through precise positioning of substrates, cofactors, and amino acids to modulate the transition-state free energy. However, the role of conformational dynamics remains poorly understood due to lack of experimental access. This shortcoming is evident with E. coli dihydrofolate reductase (DHFR), a model system for the role of protein dynamics in catalysis, for which it is unknown how the enzyme regulates the different active site environments required to facilitate proton and hydride transfer. Here, we present ligand-, temperature-, and electric-field-based perturbations during X-ray diffraction experiments that enable identification of coupled conformational changes in DHFR. We identify a global hinge motion and local networks of structural rearrangements that are engaged by substrate protonation to regulate solvent access and promote efficient catalysis. The resulting mechanism shows that DHFR's two-step catalytic mechanism is guided by a dynamic free energy landscape responsive to the state of the substrate.

High-resolution double vision of the archetypal protein tyrosine phosphatase

“…2 ). This localization of conformational flexibility to functional sites is consistent with the view that protein dynamics underlies and enables biological function (Tzeng and Kalodimos 2009; Fraser et al 2009; Wei et al 2016; Kim et al 2017; Greisman, Dalton, et al 2023).…”

“…If properly modeled, they could likely help explain functional effects of mutations and ligands (Wankowicz et al 2022), allosteric mechanisms, and other phenomena for PTP1B and other systems. Moreover, other biophysical perturbations (Keedy 2019) such as variable temperature (Fraser et al 2011;Keedy et al 2014Keedy et al , 2018Fischer 2021;Stachowski et al 2022;Sharma, Ebrahim, and Keedy 2023;Skaist Mehlman et al 2023;Greisman, Dalton, et al 2023;Thorne 2023) or pressure (Urayama, Phillips, and Gruner 2002;Guerrero et al 2023) could reveal additional, previously "hidden" conformational heterogeneity or excited states that could provide further insights into biological mechanisms for PTPs as well as other proteins. These areas represent promising avenues for future study.…”

“…Moreover, the local conformations of neighboring residues in proteins depend upon one another (Martin et al 2011; van den Bedem et al 2013; Bhattacharyya, Ghosh, and Vishveshwara 2016; Johansson and Lindorff-Larsen 2018). Importantly, even apo (unliganded) proteins are prone to sample low-occupancy conformations that change in population and contribute to function in response to molecular events (Keedy et al 2018; Wankowicz et al 2022; Greisman, Dalton, et al 2023).…”

High-resolution double vision of the allosteric phosphatase PTP1B

“…2). This localization of conformational flexibility to functional sites is consistent with the view that protein dynamics underlies and enables biological function (Tzeng & Kalodimos, 2009;Fraser et al, 2009;Wei et al, 2016;Kim et al, 2017;Greisman, Dalton et al, 2023).…”

“…Moreover, the local conformations of neighboring residues in proteins depend upon one another (Martin et al, 2011;van den Bedem et al, 2013;Bhattacharyya et al, 2015;Johansson & Lindorff-Larsen, 2018). Importantly, even apo (unliganded) proteins are prone to sample low-occupancy conformations that change in population and contribute to function in response to molecular events (Keedy et al, 2018;Wankowicz et al, 2022;Greisman, Dalton et al, 2023).…”