Heat-resistant cytosolic malate dehydrogenases (cMDHs) of thermophilic intertidal snails (genus Echinolittorina): protein underpinnings of tolerance to body temperatures reaching 55°C

Abstract

Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the MichaelisMenten constant of the cofactor NADH, K M NADH ) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners (E. malaccana and E. radiata) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata, in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases ( positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on finescale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation.

Comparative genomics reveals divergent thermal selection in warm‐ and cold‐tolerant marine mussels

“…Positively selected loci identified in this study provide opportunities to compare allele frequency patterns in multiple M. galloprovincialis populations to determine whether genes under selection for species divergence (identified in this study) are also important for local adaptation within species. Sequence data can also be used in combination with computational approaches to investigate how specific amino acid substitutions affect three‐dimensional protein structure and thermal stability, or whether these sites comprise binding regions undergoing large conformational changes during catalysis (e.g., Fields, Dong, Meng, & Somero, ; Fields et al, ; Liao et al, ; Saarman, Kober, Simison, & Pogson, ). Such data would allow a more direct assessment of the contributions of thermal selection on the evolution of species characteristics mediating present‐day distribution limits.…”

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

“…The conformational changes essential for catalytic function are also well understood, which facilitates structure-function analyses (6). Moreover, our recent work has identified regions of the protein that are key for establishing the appropriate levels of conformational flexibility, to allow the mobile regions (MRs) of the protein to undergo the changes in conformation that are essential for ligand binding and catalysis (2,3).…”

“…One involved conventional site-directed mutagenesis (SDM) followed by enzyme isolation and in vitro characterization of protein stability (rate of heat denaturation) and function (K M NADH ). The second mutagenesis technique involved MDS approaches (3). We used this in silico method to study the effects of the same substitution used in SDM on the overall structural flexibility (rmsd in backbone atom position) and residue-specific flexibility [rms fluctuation (rmsf)] of the protein.…”

Evolutionary pathways to invasive success: thermal adaptation, hybridisation and parallel evolution in a marine global invader

“…Notably, positive alpha/DoS values were identified for the thermotolerance candidate gene cMDH, corroborating that the observed amino acid differences between M. trossulus and M. galloprovincialis are likely adaptive (Fields et al 2006; Table 2-2). cMDH has been implicated, through biochemical studies, as a genetic candidate for setting species-specific thermal limits in Mytilus mussels and other marine mollusks (Mytilus mussels, Fields et al 2006; Lottia limpets, Dong and Somero 2008;Liao et al 2017). Post hoc inspections of cMDH intraspecific read alignments revealed that M. edulis is polymorphic for valine and asparagine amino acids at the functional codon 114, while M.…”

“…Determining the adaptive significance of candidate proteins and potential contributions to biological invasions, however, will require functional validation and comparative analyses of thermal physiology between species and populations before strong conclusions can be made (Dean and Thornton 2007). Sequence data can be used in combination with computational approaches to test hypotheses about how specific amino acid substitutions affect three-dimensional protein structure and thermal stability, or whether these sites comprise binding regions undergoing large conformational changes during catalysis (e.g., Fields et al 2012;Fields et al 2015;Liao et al 2017;Saarman et al 2017). Such data will have important implications for understanding the evolution of invasive characteristics that may be favoured under warming ocean conditions (Tepolt 2015).…”