AMP-activated protein kinase β-subunit requires internal motion for optimal carbohydrate binding

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• AMP-activated protein kinase (AMPK) utilizes its carbohydrate-binding module (CBM) within the β-subunit to interact with carbohydrates and glycogen.
• Two isoforms of the β-subunit, β(1)-CBM and β(2)-CBM, exist, with the muscle-specific β(2)-CBM exhibiting higher carbohydrate binding affinity than the ubiquitous β(1)-CBM.

Purpose of the Study:

• To investigate the molecular basis for the differential carbohydrate-binding affinities between the β(1)-CBM and β(2)-CBM isoforms of AMPK.
• To elucidate the role of protein dynamics and specific structural features, such as an additional threonine residue in β(2)-CBM, in modulating ligand affinity.

Main Methods:

• Comparative analysis of β(1)-CBM and β(2)-CBM using biophysical techniques to assess carbohydrate binding affinity and protein dynamics.
• Site-directed mutagenesis to investigate the functional significance of the additional threonine residue in β(2)-CBM and its insertion into β(1)-CBM.

Main Results:

• The muscle-specific β(2)-CBM demonstrates tighter carbohydrate binding compared to β(1)-CBM, despite conserved carbohydrate-contacting residues.
• An additional threonine in β(2)-CBM correlates with increased flexibility and microsecond-to-millisecond motion, particularly in a β-hairpin loop critical for carbohydrate binding.
• Mutational analysis confirmed the influence of the threonine residue on affinity and dynamics, yet indicated it is not the sole determinant of the observed differences.

Conclusions:

• Altered protein dynamics, influenced by structural variations like the additional threonine in β(2)-CBM, significantly contribute to the distinct ligand-binding affinities of the two CBM isoforms.
• Understanding these dynamics provides insights into the regulation of AMPK activity and substrate recognition.