Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

Abstract

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from -1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme's thermal responses and foster evolutionary adaptation of function.

Keywords: adaptation; cytosolic malate dehydrogenase; evolution; molecular dynamics simulations; protein evolution.

Fig. 1.

Deduced amino acid sequence alignments of 26 cMDH orthologs from marine molluscs. The substrate-binding sites, the cofactor binding sites, the active site proton acceptor site, and the residues involved in subunit–subunit interaction are shown in red circles, blue triangles, a green star, and orange diamonds, respectively. Conserved amino acid residues are highlighted in bold. Nonconservative replacements among Echinolittorina malaccana, E. radiata, Littorina keenae, and L. scutulata cMDHs are highlighted in red. Mobile regions (MRs) are highlighted in red frames.

Fig. 2.

Overall and region-specific variation in different categories of amino acids of 26 orthologs of molluscan cMDHs as a function of enzyme stability (rate of denaturation at 40 °C for genus Haliotis, Adamussium colbecki, and Laternula elliptica and at 42.5 °C for all other species). The fitted linear regression line is shown as a solid line when P < 0.05; a dashed line is used when the regression was nonsignificant. Amino acid categories were charged (DEKR), weakly hydrophobic/polar (AGNQSTHY), and hydrophobic (LVWIFMPC) (33). (A–C) Overall variation in amino acid composition throughout the protein structure, (D–F) variation in surface composition, (G–I) variation in composition of the intermediate depth region, and (J–L) variation in composition of the core region of the protein. Variation in (M–P) proline and (Q–T) glycine content in different regions of the enzyme.

Fig. 3.

Relationship between median lethal temperature (LT₅₀) and the rate of denaturation (slope of ln residual activity) of cMDH orthologs from marine molluscs. Each point represents the LT₅₀ from one species. Regression coefficients are Y = 0.00603*X – 0.279, R² = 0.720 (linear regression), and Y = −0.391 + 0.0155*X – 0.000154*X², R² = 0.866 (quadratic regression).

Fig. 4.

Three-dimensional models of a single cMDH monomer from Echinolittorina malaccana, E. radiata, Lottia austrodigitalis, L. digitalis, Chlorostoma funebralis, and C. montereyi. Red-colored ribbons identify the regions in which the increase in simulation temperature led to a significant change in structural movements [indexed by a change in rms fluctuation (rmsf) over the equilibration state (10–20 ns) greater than 0.5 Å]. Simulation temperatures were 15 °C and 57 °C for the genus Echinolittorina and 15 °C and 42 °C for the other orthologs (2). The locations of the two mobile regions are shown by dashed lines in E. malaccana cMDH monomer. The variable sites between species within each genus are indicated by yellow spheres.

Fig. 5.

Dimeric assembly of Echinolittorina malaccana cMDH. (A) The substrate-binding sites, the cofactor binding sites, the active site proton acceptor, and the residues involved in subunit–subunit interaction are shown in red, blue, mauve, and yellow spheres, respectively. The locations of the two mobile regions (residues 90–105 for MR1 and residues 230–245 for MR2) are highlighted by red shading. (B) Nonconservative replacements among E. malaccana, E. radiata, Littorina keenae, and L. scutulata cMDHs are shown in cyan spheres (residues 4, 33, 41, 48, 114, 219, and 321). The locations of the two MRs are shown by red dashed lines.

Fig. 6.

The rmsd of backbone atom positions for cMDHs at a simulation temperature of 57 °C. (A and B) Two wild-type cMDHs, Echinolittorina malaccana (wt-Em) and E. radiata (wt-Er), and two mutant forms, mut-G114S (glycine mutated to serine at site 114 for the E. malaccana ortholog) and mut-S114G (serine mutated to glycine for the E. radiata ortholog). (C) The corresponding equilibrium rmsd values (10–20 ns as shaded) (n = 10) and statistical analyses. (D and E) Two wild-type enzymes, wt-Em and Littorina keenae (wt-Lk), and two mutant enzymes, mut-P4A (proline mutated to alanine at site 4 for E. malaccana ortholog) and mut-A4P (alanine mutated to proline for L. keenae ortholog). (F) The corresponding equilibrium rmsd values (n = 5) and statistical analysis. Data are expressed as means ± SEM, and significance is denoted with asterisks: *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7.

The rms fluctuation (rmsf) for individual residues over the equilibration state (10–20 ns) of cMDHs at a simulation temperature of 57 °C. (A and B) Two wild-type enzymes, Echinolittorina malaccana (wt-Em) and E. radiata (wt-Er), and two mutant enzymes, mut-G114S (glycine mutated to serine at site 114 for the E. malaccana ortholog) and mut-S114G (serine mutated to glycine for the E. radiata ortholog) (n = 10). (C and D) Two wild-type enzymes, wt-Em and Littorina keenae (wt-Lk), and two mutant enzymes, mut-P4A (proline mutated to alanine at site 4 for the E. malaccana ortholog) and mut-A4P (alanine mutated to proline for the L. keenae ortholog) (n = 5). The locations of the two mobile regions (residues 90–105 for MR1 and residues 230–245 for MR2) are highlighted by gray shading.

Fig. 8.

Temperature dependence of the Michaelis–Menten constant for cofactor (reduced nicotinamide adenine dinucleotide) (K_M^NADH) for recombinant wild-type and mutated cMDHs. (A) Two recombinant wild types, Echinolittorina malaccana (wt-Em) and E. radiata (wt-Er), and two mutants, mut-G114S (glycine mutated to serine at site 114 for the E. malaccana ortholog) and mut-S114G (serine mutated to glycine for the E. radiata ortholog). (Inset) Arrhenius plot (ln K_M^NADH versus reciprocal temperatures in kelvin) of cMDHs. (B) Two recombinant wild types, wt-Em and Littorina keenae (wt-Lk), and two mutants, mut-P4A (proline mutated to alanine at site 4 for E. malaccana ortholog) and mut-A4P (alanine mutated to proline for L. keenae ortholog). (Inset) Arrhenius plot (ln K_M^NADH versus reciprocal temperatures in kelvin) of cMDHs. Data are expressed as means ± SEM (n = 5).

Fig. 9.

Thermal stabilities of recombinant and wild-type cMDH determined as the residual activities following incubation at 57.5 °C for different times. (A) Two recombinant wild types, Echinolittorina malaccana (wt-Em) and E. radiata (wt-Er), and two mutants, mut-G114S (glycine mutated to serine at site 114 for the E. malaccana ortholog) and mut-S114G (serine mutated to glycine for the E. radiata ortholog). (Inset) Slope of the linear regression of ln residual activity. (B) Two recombinant wild types, wt-Em and Littorina keenae (wt-Lk), and two mutants, mut-P4A (proline mutated to alanine at site 4 for the E. malaccana ortholog) and mut-A4P (alanine mutated to proline for the L. keenae ortholog). (Inset) Slope of the linear regression of ln residual activity. Significance denoted with asterisks: *P < 0.05, **P < 0.01, and ***P < 0.001. Data are expressed as means ± SEM (n = 3).