Exploring the Cold-Adaptation Mechanism of Serine Hydroxymethyltransferase by Comparative Molecular Dynamics Simulations

Abstract

Cold-adapted enzymes feature a lower thermostability and higher catalytic activity compared to their warm-active homologues, which are considered as a consequence of increased flexibility of their molecular structures. The complexity of the (thermo)stability-flexibility-activity relationship makes it difficult to define the strategies and formulate a general theory for enzyme cold adaptation. Here, the psychrophilic serine hydroxymethyltransferase (pSHMT) from Psychromonas ingrahamii and its mesophilic counterpart, mSHMT from Escherichia coli, were subjected to μs-scale multiple-replica molecular dynamics (MD) simulations to explore the cold-adaptation mechanism of the dimeric SHMT. The comparative analyses of MD trajectories reveal that pSHMT exhibits larger structural fluctuations and inter-monomer positional movements, a higher global flexibility, and considerably enhanced local flexibility involving the surface loops and active sites. The largest-amplitude motion mode of pSHMT describes the trends of inter-monomer dissociation and enlargement of the active-site cavity, whereas that of mSHMT characterizes the opposite trends. Based on the comparison of the calculated structural parameters and constructed free energy landscapes (FELs) between the two enzymes, we discuss in-depth the physicochemical principles underlying the stability-flexibility-activity relationships and conclude that (i) pSHMT adopts the global-flexibility mechanism to adapt to the cold environment and, (ii) optimizing the protein-solvent interactions and loosening the inter-monomer association are the main strategies for pSHMT to enhance its flexibility.

Keywords: cold adaptation; free energy landscape; molecular dynamics simulation; protein-solvent interactions; stability-flexibility-activity relationships.

Figure 1

Cartoon representations of the crystal structures of two differently temperature-adapted serine hydroxymethyltransferases (SHMT) and their backbone superposition. (A,B) The dimeric forms of the mesophilic SHMT (mSHMT) from Escherichia coli (PDB ID: 1DFO [18]) and the psychrophilic SHMT (pSHMT) from Psychromonas ingrahamii (PDB ID: 4P3M [19]), respectively. (C) Backbone superposition of the two structures. (D) The monomeric form of mSHMT. The missing residues in the crystal structures were modeled as described in Section 4.1. In (A,B), the monomer-A and monomer-B are colored green and orange, respectively; the active-site components of the monomer-A, i.e., the “floor”, “walls”, and “roof” are colored cyan, blue, and magenta, respectively. In (C), the backbones of mSHMT and pSHMT are colored red and green, respectively. In (D), The N-terminal arm, large domain, small domain, and inter-domain linkers are colored blue, green, red, and yellow, respectively.

Figure 2

Time evolution of the C_α root mean square deviation (RMSD) values of mSHMT and pSHMT with respect to their respective starting structures calculated from the 10 MD simulation replicas (r1-10). (A) mSHMT. (B) pSHMT.

Figure 3

Per-residue C_α Root-mean-square fluctuation (RMSF) profiles of mSHMT (red line) and pSHMT (green line). Residues that constitute the “floor” (residues 197–204 and 213–242), “walls” (inner “wall”: residues 97–110 from the monomer-A and 258–264 from the monomer-B; outer “wall”: residues 174–182 from the monomer-A), and “roof” (residues 118–133) of the active site in the monomer-A are indicated above the horizontal axis by line segments colored in cyan, blue, and magenta, respectively.

Figure 4

Eigenvalues of the first 30 eigenvectors (main plot) and cumulative contribution of all eigenvectors to the total mean square fluctuations (inset plot) obtained from essential dynamics analyses of the single joined equilibrium MD trajectories of mSHMT (red line) and pSHMT (green line).

Figure 5

Porcupine plots showing the largest-amplitude collective motion along the first eigenvector. (A,B) The most significant motion modes of mSHMT and pSHMT, respectively. In a dimer, the monomer-A is rendered in cartoon representation, with the N-terminal arm, large domain, and small domain colored blue, green, and red, respectively; the monomer-B is rendered in ribbon and colored cyan. In the monomer-B, a small segment (residues 258–264) that participates in the formation of the inner “wall” of monomer-A’s active-site cavity is colored orange. In the monomer-A, the residue numbers for the starting and ending residues of the segments participating in the formation of the active-site cavity are labeled.

Figure 6

Constructed free energy landscapes (FELs) of mSHMT and pSHMT using the reaction coordinates as the projection of the joined equilibrium MD trajectory onto an essential subspace spanned by eigenvectors 1 and 2. (A) FEL of the mSHMT. (B) FEL of the pSHMT. The color bar represents the relative free energy value in kJ/mol.