Elucidating the preference of dimeric over monomeric form for thermal stability of Thermus thermophilus isopropylmalate dehydrogenase: A molecular dynamics perspective
Graphical abstract
Introduction
Temperature has a strong influence on an organism’s evolution through the optimization of their protein repertoire. Thermophiles thrive at high temperature and their enzymes have significant relevance to industrial applications [[1], [2], [3]]. The protein repertoire of a thermophilic organism exhibit temperature adaptive factors making them structurally stable to temperature [4]. Studying proteins from thermophiles helps in understanding the factors contributing to its stability and thereby function at high temperature. Many reactions are carried out at high temperature to increase the solubility of a substrate or to reduce the risk of contaminations, thus is of prime importance in industrial production [2,5]. Several proteins of thermophiles are oligomers [6,7]. Thus, the link between the protein oligomerization and thermal stability needs a comprehensive investigation [8].
A simplest oligomer is a dimer. The functional form of IPMDH is dimeric in conformation [9] and can retain three-dimensional structure at 337 K [10]. It is one of the most widely studied proteins to understand the factors that influence thermostability [[10], [11], [12], [13], [14], [15], [16]]. Site-directed mutagenesis studies have allowed the identification of mutants to improve the thermal stability of the protein [10,11,[17], [18], [19]]. To elucidate the structure, stability relationship, chimeric IPMDH was investigated [15,20]. Crystallographic structures of the enzyme from T. thermophilus have been determined for wild type (PDB ID: 1IPD) at 2.2 Å resolution [9]. It is a homodimer protein with two domains in each subunit of 345 residues. Domain 1 consists of residue range 1–99, 252–345 and remaining forms, domain 2. The similar trend is followed in the domains of subunit II [9]. Both domains in subunits are similar in their folding topologies and conformations. Crystallographic structure of the dimer protein suggests that domain 1 of each subunit consists of seven α-helices and five β-strands. All β-strands are involved in forming central β-sheet. Three helices are continuously formed from I285 to A345. Domain 2 contains four α-helices and seven β-strands. Thus, a subunit consists of total eleven α-helices and twelve β-strands [9] (Fig. 1).
The subunits are in contact mainly at four regions, A) loop containing residues 116–118 with 116′-118′, B) 216–225 with 237′-250′, C) 237–250 with 216′-225′, and D) 150–158 with 150′-158’ (Fig. 1A and B, Fig. S1). The symbol (’) represents the residue from another subunit. Imada et al. [9] proposed that subunit-subunit interaction is necessary for its thermal stability. Mutations at the dimer interface depict a decrease in thermal stability of the protein [5,7]. G240A and L246E/V249 M located at one of the major regions of subunit contact decrease the protein’s thermal stability [16]. Crystallographic studies provide static conformation, although valuable are often affected by the different crystallization conditions, mutations and temperatures at which data is collected. The molecular basis with which the preference of dimerization has an influence on thermal stability of the protein remains poorly understood and demands extensive analysis.
Here, we made an attempt to study the relation between dimerization with protein’s thermal stability by studying the dynamics of a subunit in the presence and absence of another subunit hereafter referred to as SS and SA, respectively with the same initial structure downloaded from Protein Data Bank (PDB) (PDB ID: 1IPD). The temperature at which the respective simulations are performed is in subscript. Extending our ongoing efforts in understanding the factors contributing to the thermal stability of the protein [21], this is the first classical method applied on the protein to gain an insight on the preference of dimerization for the thermal stability of the protein. The study allows comparing the need of another subunit with the change in temperature starting from the same initial experimental structure, keeping rest of the conditions similar before denaturing temperature. Further, ANM inspired MD has advantages over traditional MDs in its ability to predict large scale fluctuations resulting from the collective motions of -domains. The detailed analysis shall provide insights on explaining the influence of oligomerization on the thermal stability of a protein.
Section snippets
Materials and methods
Atomic coordinates of monomer in the asymmetric unit of resolution 2.2 Å were downloaded from the PDB (PDB ID: 1IPD) [9]. Functional dimer of wild type was generated using symmetry equivalent molecules. The α-helices and β-strands in the SA and SS were coded as H and B, respectively. It is followed by the digit 1 or 2 to mark its presence in domain 1 or 2, respectively. The third digit in code corresponds to its sequential presence in the domain (Fig. 1C and Table S1).
All atom optimized
Results and discussion
IPMDH is functionally a homodimer. To scrutinize the need for another subunit to enhance the thermal stability, we perceived how the dynamic properties of SS and SA changes with the change in temperature before denaturation using the initial conformation as the crystallographic structure (PDB: 1IPD).
Structural stability of SS and SA at 300 K and 337 K: Monitoring the structural stability of SS and SA at 300 K and 337 K by RMSD of Cα atoms (1–345) with respect to the asymmetric unit of PDB ID: 1IPD
Conclusions
The study investigates the preference of dimeric over monomeric form of IPMDH for thermal stability by comparing the structural stability of a subunit in the presence (dimer) and absence of another subunit (monomer) with an increase in temperature and is in good agreement with the experimental findings known so far.
The significantly high RMSD with respect to the initial reference structure, more SASA, higher flexibility and more exposure of hydrophobic residues to solvent in SA at high
Declaration of competing interest
The authors have declared that no competing interests exist.
Acknowledgements
RS thanks Department of Biotechnology (DBT)-BioCARe research grant (BT/PR18249/BIC/101/390/2016) for the financial support. IICT manuscript communication number: IICT/Pubs./2019/082. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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