Capture and quality control mechanisms for adenosine-5'-triphosphate binding

Area of Science:

• Biochemistry
• Molecular Biology
• Computational Chemistry

Background:

• Nucleotidylyl transferase superfamily enzymes catalyze crucial biological reactions.
• Aminoacyl-tRNA synthetases (aaRSs) are the largest and most studied group within this superfamily.
• Efficient and accurate ATP binding is essential for aaRS function.

Purpose of the Study:

• To elucidate the free energy landscape of ATP selection and binding in class I aaRSs.
• To identify key intermediate states and catalytic mechanisms involved in ATP binding.
• To understand the role of specific residues, like the conserved histidine, in ATP binding and selectivity.

Main Methods:

• Metadynamics simulations to calculate the free energy landscape of ATP binding.
• Site-directed mutagenesis to probe the function of key amino acid residues.
• Fluorescence spectroscopy to experimentally validate simulation findings and binding affinities.

Main Results:

• Identified distinct intermediate states in the ATP binding process, including encounter complexes and a nucleoside binding state.
• Revealed a 'fly casting' mechanism for initial ATP triphosphate binding and base-stacking interactions mediated by a conserved histidine for nucleoside binding.
• Demonstrated that mutation of the conserved histidine significantly reduces ATP binding affinity.
• Discovered an intermediate quality control state crucial for ATP selectivity over other nucleoside triphosphates.

Conclusions:

• The study provides a detailed mechanistic understanding of ATP binding and selection in aaRSs.
• The identified quality control mechanism is likely conserved across the nucleotidylyl transferase superfamily.
• Computational simulations combined with experimental validation offer powerful insights into enzyme catalysis.