Temperature as a modulator of allosteric motions and crosstalk in mesophilic and thermophilic enzymes

Abstract

Mesophilic and thermophilic enzyme counterparts are often studied to understand how proteins function under harsh conditions. To function well outside of standard temperature ranges, thermophiles often tightly regulate their structural ensemble through intra-protein communication (via allostery) and altered interactions with ligands. It has also become apparent in recent years that the enhancement or diminution of allosteric crosstalk can be temperature-dependent and distinguish thermophilic enzymes from their mesophilic paralogs. Since most studies of allostery utilize chemical modifications from pH, mutations, or ligands, the impact of temperature on allosteric function is comparatively understudied. Here, we discuss the biophysical methods, as well as critical case studies, that dissect temperature-dependent function of mesophilic-thermophilic enzyme pairs and their allosteric regulation across a range of temperatures.

Keywords: NMR; allostery; kinetics; temperature; thermodynamics; thermophiles.

FIGURE 1

Kinetic and thermodynamic aspects of protein conformational equilibria are temperature-sensitive. (A) Temperature dependence of a Boltzmann distribution showing increased degeneracy at higher temperatures. Created with BioRender. (B) Protein folding landscapes rely on a delicate balance of enthalpy and entropy that are sensitive to temperature. Created with BioRender. (C) Many biological processes have distinct temporal and energetic requirements that define protein conformational states. Variable temperatures can skew energetic barriers that rewire protein motions to directly influence biochemical mechanisms.

FIGURE 2

(A) X-ray crystallographic overlay of Proteinase K structures collected at 100 K (PDB ID:7LTD, blue) and 363 K (PDB ID:8SOU, orange) highlighting temperature-dependent differences in the backbone conformation and ligand binding pocket. A Ca²⁺ binding pocket in Proteinase K, showing four distinct binding locations at 363 K, but only a single binding mode at 100 K. (B) X-ray crystallographic overlay of Proteinase K structures collected at 313 K (PDB ID: 8SOG, red) and 363 K (PDB ID: 8SOU, blue) showing differences the backbone and side chain conformation, as well as ligand binding pocket. Ca²⁺ binding locations once again vary at two elevated temperatures and are represented in cyan at 313 K and orange at 363 K.

FIGURE 3

Overlay of ¹H-¹³CH₃ methyl-ILV NMR spectra collected at 303 K (red), 323 K (blue), and 343 K (gold) depicting temperature-dependent variations in exchange regime. Arrows following chemical shift trajectories bifurcate when minor populations of a given resonance become visible. Here, elevated temperatures more clearly resolve multiple conformations within the HisF subunit of IGPS. Resonances labeled in green were unassigned in the original data presented by Loria and coworkers in Front. Mol. Biosci. 2017.

FIGURE 4

(A) Differences in Eigenvector Centrality within IGPS, intrinsic to the binding of PRFAR (holo enzyme) at 303 K (left) or 323 K (right) and without effector binding (apo enzyme) at the elevated temperature of 323 K (center). Communication pathways induced by PRFAR at 303 K are recapitulated solely due to the effect of temperature in the apo enzyme at 323 K. (B) Comparative transmission of distinct mutual information pathways linking the PRFAR binding site to the active site (hG50 in HisH, red sphere). Areas of secondary structure involved in communication pathways are separately highlighted as solvent exposed amino acids (yellow, “external”) and buried (green, “internal”). Similar trends are observed in for the holo enzyme at 303 K and apo enzyme at 323 K when compared to apo IGPS (303 K), specifically a transition from internal pathways to external communication due to effector binding and/or temperature increase. Figure was reproduced from Maschietto et al. (2023), Nature Communications, under a Creative Commons Attribution 4.0 International License.