3D Variability Analysis Reveals a Hidden Conformational Change Controlling Ammonia Transport in Human Asparagine...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Understanding enzyme dynamics is key to elucidating catalytic mechanisms.
• Advanced imaging techniques like cryo-electron microscopy (cryo-EM) reveal conformational changes.
• 3D variability analysis (3DVA) aids in studying these dynamic processes.

Purpose of the Study:

• To investigate the role of enzyme motions in catalytic function.
• To identify the function of the Arg-142 side chain in human asparagine synthetase (ASNS).
• To explore how conformational changes regulate enzyme activity.

Main Methods:

• 3D variability analysis (3DVA) of cryo-EM data for wild-type (WT) human ASNS.
• Experimental assessment using the R142I variant.
• Molecular dynamics (MD) simulations of ASNS monomer and ternary complex.

Main Results:

• 3DVA identified Arg-142 as a gate controlling ammonia access to an intramolecular tunnel.
• MD simulations confirmed residue fluctuations and the separation of gating from tunnel formation.
• Experimental data showed the R142I variant's altered glutamine-dependent synthetase activity.

Conclusions:

• Arg-142 acts as a gate regulating ammonia translocation in ASNS.
• The combination of 3DVA and MD simulations is a powerful approach for generating testable hypotheses.
• Conformational changes in buried side chains can regulate enzyme function.