A comparative study of cold- and warm-adapted Endonucleases A using sequence analyses and molecular dynamics...

Area of Science:

• Biophysics
• Structural Biology
• Enzymology

Background:

• Investigating thermal adaptation in enzymes like endonucleases A (EndA) is crucial for understanding protein function across different temperatures.
• Static X-ray structures of psychrophilic Aliivibrio salmonicida EndA (VsEndA) and mesophilic Vibrio cholera EndA (VcEndA) were insufficient to explain their thermal adaptation.
• Molecular Dynamics (MD) simulations offer a powerful approach to explore protein dynamics and uncover determinants of thermal adaptation.

Purpose of the Study:

• To compare the biophysical properties and molecular determinants of thermal adaptation in VsEndA and VcEndA.
• To utilize MD simulations to identify structural and dynamical differences between the two enzymes.
• To pinpoint specific amino acid substitutions contributing to the distinct thermal adaptation of EndA variants.

Main Methods:

• Comparative analysis of static X-ray structures.
• Nanosecond-scale Molecular Dynamics (MD) simulations of VsEndA and VcEndA.
• Analysis of MD-based protein structure networks and multiple sequence alignment.
• MD simulations of a VsEndA mutant variant with introduced electrostatic interactions.

Main Results:

• MD simulations did not reveal significant overall flexibility differences between the cold-adapted and mesophilic variants on the nanosecond timescale.
• A more rigid C-terminal region in VcEndA was identified, attributed to electrostatic interactions and hydrogen bonds.
• Three key amino acid substitutions were identified, including T120V near the catalytic residue H80, impacting interactions within the Mg2+ ion coordination sphere.

Conclusions:

• The study highlights the importance of subtle structural and dynamic features, rather than global flexibility, in enzyme thermal adaptation.
• The identified rigid C-terminal region in VcEndA and the T120V substitution are significant findings for understanding EndA thermal adaptation.
• The T120V substitution presents a promising target for future experimental mutagenesis studies to validate its role in enzyme function and adaptation.