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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.

The entry of human immunodeficiency virus type I (HIV-1) into host cells is initiated by binding to the cell-surface receptor CD4, which induces a conformational transition of the envelope (Env) glycoprotein gp120 from the closed, unliganded state to the open, CD4-bound state. Despite many available structures in these two states, detailed aspects on the dynamics and thermodynamics of gp120 remain elusive. Here, we performed microsecond-scale (μs-scale) multiple-replica molecular dynamics (MD) simulations to explore the differences in the conformational dynamics, protein motions, and thermodynamics between the unliganded and CD4-bound/complexed forms of gp120. Comparative analyses of MD trajectories reveal that CD4 binding promotes the structural deviations/changes and conformational flexibility, loosens the structural packing, and complicates the molecular motions of gp120. Comparison of the constructed free energy landscapes (FELs) reveals that the CD4-complexed gp120 has more conformational substates, larger conformational entropy, and lower thermostability than the unliganded form. Therefore, the unliganded conformation represents a structurally and energetically stable "ground state" for the full-length gp120. The observed great increase in the mobility of V1/V2 and V3 along with their more versatile movement directions in the CD4-bound gp120 compared to the unliganded form suggests that their orientations with respect to each other and to the structural core determine the differences in the conformational dynamics and thermodynamics between the two gp120 forms. The results presented here provide a basis by which to better understand the functional and immunological properties of gp120 and, furthermore, to deploy appropriate strategies for the development of anti-HIV-1 drugs or vaccines.

Molecular dynamics (MD) simulations of a subtilisin-like serine protease VPR from the psychrophilic marine bacterium Vibrio sp. PA-44 and its mesophilic homologue, proteinase K (PRK), have been performed for 20 ns at four different temperatures (300, 373, 473, and 573 K). The comparative analyses of MD trajectories reveal that at almost all temperatures, VPR exhibits greater structural fluctuations/deviations, more unstable regular secondary structural elements, and higher global flexibility than PRK. Although these two proteases follow similar unfolding pathways at high temperatures, VPR initiates unfolding at a lower temperature and unfolds faster at the same high temperatures than PRK. These observations collectively indicate that VPR is less stable and more heat-labile than PRK. Analyses of the structural/geometrical properties reveal that, when compared to PRK, VPR has larger radius of gyration (Rg), less intramolecular contacts and hydrogen bonds (HBs), more protein-solvent HBs, and smaller burial of nonpolar area and larger exposure of polar area. These suggest that the increased flexibility of VPR would be most likely caused by its reduced intramolecular interactions and more favourable protein-solvent interactions arising from the larger exposure of the polar area, whereas the enhanced stability of PRK could be ascribed to its increased intramolecular interactions arising from the better optimized hydrophobicity. The factors responsible for the significant differences in local flexibility between these two proteases were also analyzed and ascertained. This study provides insights into molecular basis of thermostability of homologous serine proteases adapted to different temperatures.