Mechanism of deep-sea fish α-actin pressure tolerance investigated by molecular dynamics simulations

Abstract

The pressure tolerance of monomeric α-actin proteins from the deep-sea fish Coryphaenoides armatus and C. yaquinae was compared to that of non-deep-sea fish C. acrolepis, carp, and rabbit/human/chicken actins using molecular dynamics simulations at 0.1 and 60 MPa. The amino acid sequences of actins are highly conserved across a variety of species. The actins from C. armatus and C. yaquinae have the specific substitutions Q137K/V54A and Q137K/L67P, respectively, relative to C. acrolepis, and are pressure tolerant to depths of at least 6000 m. At high pressure, we observed significant changes in the salt bridge patterns in deep-sea fish actins, and these changes are expected to stabilize ATP binding and subdomain arrangement. Salt bridges between ATP and K137, formed in deep-sea fish actins, are expected to stabilize ATP binding even at high pressure. At high pressure, deep-sea fish actins also formed a greater total number of salt bridges than non-deep-sea fish actins owing to the formation of inter-helix/strand and inter-subdomain salt bridges. Free energy analysis suggests that deep-sea fish actins are stabilized to a greater degree by the conformational energy decrease associated with pressure effect.

Figure 1. Structure of monomeric actin.

(A) Subdomain arrangement. Subdomains 1, 2, 3 and 4 are shown in cyan, red, yellow and green, respectively. The pink sphere represents Mg²⁺ at the active site. (B) Positions of substituted residues in C. yaquinae actin as compared to rabbit/chicken actin. The residues shown in red and cyan in the licorice model represent the specific substitutions in deep-sea fish actins and those of terrestrial animals and shallow-water fish species, respectively. (C) Chemical formula of ATP. Oxygen atoms in the phosphate tail of ATP are distinguished by α, β, and γ.

Figure 2. The root mean-square fluctuation (RMSF) per residue at 60 MPa.

The RMSF was calculated by best-fitting the backbone heavy atoms of each snapshot to the average structure. Secondary structure and subdomain assignments are also shown.

Figure 3. Salt bridge and hydrophobic interactions in actin.

The salt bridges (A) between secondary structures and (B) between subdomains in Yaq at 60 MPa. The residues that form salt bridges with a formation rate of more than 0.5 are shown in Yaq at 60 MPa. Red and blue represent acidic and basic amino acids, respectively. (C) Hydrophobic interactions involving specific substituted residues in Ac1 actin. Red broken lines indicate the hydrophobic interaction. Residues 54 and 67 are different in the actins of deep-sea fish and other species.

Figure 4. Arrangement of the water molecule expected to initiate nucleophilic attack on the γ-phosphate of ATP.

The arrangement of non-deep-sea fish actins (A) and deep-sea fish actins (B). Green spheres show water molecules expected to be nucleophilic water for ATP hydrolysis. Red spheres indicate the water molecules coordinated to Mg²⁺ and those bridging the expected nucleophilic water and H161 with hydrogen bonds. Black dotted lines show typical hydrogen bonds formed during the MD simulation. Angle θ and distance d _Nu are defined by O^β-P^γ-O^w and P^γ-O^w, respectively, where O^w represents the oxygen of the expected nucleophilic water (see Figure 1C for the definition of the other atoms).

Figure 5. Spatial distribution of expected nucleophilic water.

Distribution of expected nucleophilic water as a function of angle θ and distance d _Nu, as defined in the legend for Figure 4, converted as free energy scale at (A) 0.1 and (B) 60 MPa. A water molecule having the minimum d _Nu value and a θ greater than 109.3° was assigned as the expected nucleophilic water in each simulation snapshot. The free energy was shown as the relative value against the minimum free energy in k _B T.