Structural flexibility and protein adaptation to temperature: Molecular dynamics analysis of malate dehydrogenases of marine molluscs

Abstract

Orthologous proteins of species adapted to different temperatures exhibit differences in stability and function that are interpreted to reflect adaptive variation in structural "flexibility." However, quantifying flexibility and comparing flexibility across proteins has remained a challenge. To address this issue, we examined temperature effects on cytosolic malate dehydrogenase (cMDH) orthologs from differently thermally adapted congeners of five genera of marine molluscs whose field body temperatures span a range of ∼60 °C. We describe consistent patterns of convergent evolution in adaptation of function [temperature effects on K_M of cofactor (NADH)] and structural stability (rate of heat denaturation of activity). To determine how these differences depend on flexibilities of overall structure and of regions known to be important in binding and catalysis, we performed molecular dynamics simulation (MDS) analyses. MDS analyses revealed a significant negative correlation between adaptation temperature and heat-induced increase of backbone atom movements [root mean square deviation (rmsd) of main-chain atoms]. Root mean square fluctuations (RMSFs) of movement by individual amino acid residues varied across the sequence in a qualitatively similar pattern among orthologs. Regions of sequence involved in ligand binding and catalysis-termed mobile regions 1 and 2 (MR1 and MR2), respectively-showed the largest values for RMSF. Heat-induced changes in RMSF values across the sequence and, importantly, in MR1 and MR2 were greatest in cold-adapted species. MDS methods are shown to provide powerful tools for examining adaptation of enzymes by providing a quantitative index of protein flexibility and identifying sequence regions where adaptive change in flexibility occurs.

Keywords: adaptation; amino acid sequence; evolution; malate dehydrogenase; molecular dynamics simulations.

Fig. 1.

Effects of temperature on cofactor binding affinity (K_M^NADH) (Left) and time-dependent loss of activity (Right) for 12 orthologs of cMDH from five genera of marine molluscs (mean ± SEM, n = 5, genus Echinolittorina; mean ± SEM, n = 3, genus Nerita; mean ± SEM, n = 3, genus Lottia; mean ± SEM, n = 3, genus Littorina; mean ± SEM, n = 3, genus Chlorostoma). Approximate upper lethal temperatures for the species are E. malaccana (55 °C), Echinolittorina radiata (52 °C), Nerita albicilla (50 °C), Nerita yoldii (50 °C), L. austrodigitalis (41 °C), L. digitalis (40 °C), Littorina keenae (48 °C), Littorina scutulata (45 °C), C. rugosa (∼45 °C), C. funebralis (42 °C), C. brunnea (36 °C), and C. montereyi (36 °C). (See Fig. S1 for biogeographic information and sources of thermal tolerance data. Slopes of rates of loss of activity are given in Table S1).

Fig. 2.

MDS analysis of backbone atom movements in cMDHs. (A and B) The rmsd of backbone atom positions for cMDHs from Echinolittorina malaccana, E. radiata, C. funebralis, C. brunnea, and C. montereyi at simulation temperatures of 15 and 42 °C (n = 3). (C) The ΔRMSD (the difference of rmsd value between 42 and 15 °C) over the equilibration state (10–20 ns) vs. lethal temperature (LT₅₀) for cMDH orthologs of 11 species of molluscs. (D) The ΔRMSD vs. the rate of thermal denaturation for 11 cMDH orthologs. Species are numbered as follows: 1, E. malaccana; 2, E. radiata; 3, N. albicilla; 4, N. yoldii; 5, L. keenae; 6, L. scutulata; 7, L. austrodigitalis; 8, L. digitalis; 9, C. funebralis; 10, C. brunnea; and 11, C. montereyi. The relationship between ΔRMSD and LT₅₀ or rate of thermal denaturation was analyzed by a least-squares linear regression analysis model.

Fig. 3.

MDS analysis of side chain movements (RMSF) in molluscan cMDHs. RMSF spectra for congeners of Echinolittorina (A) and Chlorostoma (B) at simulation temperatures of 15 and 42 °C (n = 3). Values for average RMSF across the full sequence are given in Table S1. (C and D) Number of residues showing a change in RMSF (ΔRMSF) > 0.5 Å vs. the species’ lethal temperature (LT₅₀) (C) and the rate of thermal denaturation (D) for the cMDH orthologs. (E and F) Number of residues occurring in MR regions (residues 90–105 and 230–245) showing ΔRMSF > 0.5 Å vs. LT₅₀ (E) and the rate of thermal denaturation (F) for the cMDH orthologs. The numbers assigned to each species are given in the Fig. 2 legend. The relationships between the number of residues showing significant change and the LT₅₀ or rate of thermal denaturation were analyzed by least-squares linear regression.