Unravelling the Adaptation Mechanisms to High Pressure in Proteins

Abstract

Life is thought to have appeared in the depth of the sea under high hydrostatic pressure. Nowadays, it is known that the deep biosphere hosts a myriad of life forms thriving under high-pressure conditions. However, the evolutionary mechanisms leading to their adaptation are still not known. Here, we show the molecular bases of these mechanisms through a joint structural and dynamical study of two orthologous proteins. We observed that pressure adaptation involves the decoupling of protein-water dynamics and the elimination of cavities in the protein core. This is achieved by rearranging the charged residues on the protein surface and using bulkier hydrophobic residues in the core. These findings will be the starting point in the search for a complete genomic model explaining high-pressure adaptation.

Keywords: high pressure adaptation; neutron scattering; origins of life; protein dynamics.

Figure 1

Crystal structures of Tba PMI (a) and Tko PMI (b) with labelled substituted residues for comparison. Residues are color-coded for polarity (red for acidic, blue for basic, green for polar, and yellow for hydrophobic), and the active site is also shown (details in Figure S7).

Figure 2

Internal cavities in the two proteins located near Ile-Val substitutions in the protein core (a,b) and at the dimer interface (c,d) after the relaxation of the crystal structures by MD at 310 K and 1 bar.

Figure 3

Scattering curves for Tba PMI at 150 bar and at some representative temperatures, with the corresponding two-state model fits. The decrease of the elastic intensity with temperature is expected, as more motions enter the time window of the experiment and the involved atoms scatter neutrons inelastically.

Figure 4

Total MSD (full circles) and MSD into the single wells ($\Delta x_0^2$, open circles) for Tba PMI (a) and Tko PMI (b), lines are a guide to the eye. The absolute value of the MSD reports on the amplitude of hydrogen atoms motion, while the slope of the curve as a function of temperature indicates how much energy is necessary to increase said amplitude, i.e., the resilience of the protein [32].

Figure 5

Temperature-independent parameters extracted from the two-state model as a function of pressure for Tba PMI (black symbols) and Tko PMI (red symbols): distance between the two wells (d, (a)), enthalpy ($\Delta H$, (b)) and entropy ($\Delta S$, (c)) difference, lines are a guide to the eye. (b) also contains a pictorial representation of the model showing how the different parameters influence the shape of a proteins’ energy landscape.

Figure 6

Fit example of Tba PMI at 1 bar and 286 K, at a q value of 1.14 Å⁻¹. Black circles represent the corrected data, the two Lorentzian contributions owing to localized motions and jump–diffusion are shown respectively as blue and green solid lines, and the total fit is shown as a red solid line. The resolution function is also shown. Error bars are smaller than the symbol size.

Figure 7

Natural logarithm of the broad component HWHM as a function of inverse temperature for Tba PMI (a) and Tko PMI (b) at all pressure values. Lines are linear fittings assuming an Arrhenius behaviour. Activation energy values are reported on the figure color-coded with the plots. Mean jump length from the Hall–Ross model as a function of temperature and pressure for Tba PMI (c) and Tko PMI (d). Natural logarithm of the residence time as a function of inverse temperature at all pressure values. Lines are fits to the Arrhenius law ((e), Tba PMI) or to the Vogel–Fulcher–Tamman law ((f), Tko PMI). Values for the activation energy (Tba PMI) and the fragility index (Tko PMI) are reported on the figure color-coded with the plots. Values of confinement radius R for Tba PMI (g) and Tko PMI (h).

Figure 8

Representation of internal (magenta) and solvent-accessible (cyan) cavities for Tba PMI (a–c) and Tko PMI (d–f) at different T and P conditions. Structures are represented in cartoons, and substituted residues are evidenced in sticks (with the corresponding part of the ribbon coloured for residue type, blue for basic, red for acidic, green for polar and yellow for hydrophobic). Red arrows follow the evolution of some cavities, and the opposite behaviour of the internal cavities in the dimer interface of the two proteins is highlighted by red circles. Another red circle in (f) also shows how the ligand pocket in one monomer of Tko PMI has actually become a channel from side to side under extreme conditions.

Figure 9

(a) sequence alignment of the two proteins, substitutions are highlighted (red for acidic, blue for basic, green for polar and yellow for hydrophobic residues). (b) schematic representation of the two proteins and effect of high pressure on them. (c) cartoon representation of Tba PMI, substitutions are represented in sticks with the same color-code as (b). (d) vertical cut on the surface representation of Tba PMI, highlighting the ligand pocket.