Temperature-Resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions

Abstract

Protein functions are temperature-dependent, but protein structures are usually solved at a single (often low) temperature because of limitations on the conditions of crystal growth or protein vitrification. Here we demonstrate the feasibility of solving cryo-EM structures of proteins vitrified at high temperatures, solve 12 structures of an archaeal ketol-acid reductoisomerase (KARI) vitrified at 4−70 °C, and show that structures of both the Mg 2+ form (KARI:2Mg 2+ ) and its ternary complex (KARI:2Mg 2+ :NADH:inhibitor) are temperature-dependent in correlation with the temperature dependence of enzyme activity. Furthermore, structural analyses led to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects and to capture of temperature-resolved intermediates of the temperature-induced conformational change. The results also suggest that it is preferable to solve cryo-EM structures of protein complexes at functional temperatures. These studies should greatly expand the landscapes of protein structure−function relationships and enhance the mechanistic analysis of enzymatic functions. Communication pubs.acs.org/JACS

Advances in methods for atomic resolution macromolecular structure determination

“…In the decade since this key observation was made, a number of additional cases have demonstrated that structural information derived from data collected closer to physiological temperature (or across a range of temperatures) can provide a wealth of information about the conformational ensemble beyond a single static structure 115 , 126 – 128 . As noted above, similar ideas are emerging in electron microscopy 94 .…”

“…An emerging frontier in cryo-EM data interpretation is the analysis of energetic landscapes and the dissection of structural heterogeneity 94 . In a cryo-EM data set with a large number of particles, the equilibrium ensemble of conformational states should be statistically represented, offering the ability to model different conformations of a molecule or complex from a single sample if one can correctly classify the particles into groups sharing the same conformational state.…”

Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM

“…This was only possible because the studied processes were slower than the time required to prepare a cryo-EM sample grid by the standard method of manual application, followed by automated blotting and plunge-freezing [14][15][16][17] . Dynamic structural states could also be resolved by freeze-trapping samples, pre-incubated at different temperatures 18 . However, both of these techniques are applicable to a small subset of biochemical reactions that have slow kinetics or show substantial temperature sensitivity, and do not enrich short-lived intermediates.…”

Annealing synchronizes the 70S ribosome into a minimum-energy conformation

“…2A). Researchers recently developed temperature-resolved cryo-EM to study temperature-sensitive proteins, such as transient receptor potential vanilloid-3 (TRPV3) and ketol-acid reductoisomerase (KARI), at optimal temperatures (27, 28). We optimized sample preparation, such as by consistent high-temperature control and efficient prevention of ice contamination, and found these procedures useful for obtaining ideal cryo-EM grids (fig.…”