Methyl transfer by substrate signaling from a knotted protein fold

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Enzymology

Background:

• Knotted proteins possess restricted conformational flexibility compared to unknotted counterparts.
• The dynamic capabilities of knotted proteins in mediating long-range communication between binding sites remain largely unexplored.
• TrmD, a bacterial methyltransferase, utilizes a unique knotted fold for methyl transfer to G37-tRNA, producing m¹G37-tRNA, vital for protein synthesis.

Purpose of the Study:

• To investigate the role of the TrmD protein knot in its catalytic activity and dynamics.
• To understand how the TrmD knot responds to substrate binding, specifically S-adenosyl methionine (AdoMet).
• To elucidate the mechanism by which the knot facilitates communication and stabilizes interactions within the active site.

Main Methods:

• Integrated structural analysis of TrmD.
• Kinetic studies to assess enzyme activity and substrate interactions.
• Computational modeling to analyze protein dynamics and energy propagation.

Main Results:

• The structurally constrained TrmD knot is essential for its methyltransferase catalytic function.
• The TrmD knot exhibits complex internal movements that are modulated by AdoMet binding.
• AdoMet binding energy is propagated through the knot, stabilizing tRNA binding and promoting active site assembly.

Conclusions:

• Protein knots are not merely structural constraints but active participants in enzyme catalysis.
• The TrmD knot functions as an organized structure that captures substrate binding free energy.
• This energy capture mechanism facilitates catalysis by stabilizing interactions critical for enzyme function.