Identification and function of conformational dynamics in the multidomain GTPase dynamin

Area of Science:

• Cell biology
• Biochemistry
• Structural biology

Background:

• Vesicle release during endocytosis depends on membrane fission.
• The large GTPase dynamin catalyzes this essential cellular process.
• Dynamin's five domains coordinate its mechanochemical functions.

Purpose of the Study:

• To investigate nucleotide- and membrane-binding-dependent conformational changes in dynamin.
• To identify allosteric mechanisms regulating dynamin's function.
• To understand the role of the pleckstrin homology domain (PHD) in dynamin-catalyzed membrane fission.

Main Methods:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes.
• Förster resonance energy transfer (FRET) to analyze domain movements.
• Engineering dynamin constructs (cross-linking, mutagenesis) to lock domain conformations.

Main Results:

• HDX-MS revealed global conformational shifts upon nucleotide and membrane binding.
• An allosteric relay in the dynamin stalk domain was identified.
• FRET confirmed large movements of the PHD between closed and open states.
• Engineered dynamin constructs demonstrated PHD movement as a regulatory switch for self-assembly, membrane binding, and fission.
• A centronuclear myopathy mutation (S619L) impaired this PHD conformational switch.

Conclusions:

• Dynamin's PHD acts as a conformational switch, regulating its assembly and membrane fission activity.
• Coordinated conformational changes link membrane binding, oligomerization, and GTPase activity in dynamin.
• The S619L mutation's impact on the PHD switch highlights its physiological importance in dynamin function and disease.