Cyclostreptin and microtubules: is a low-affinity binding site required?

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Cyclostreptin (CS) is a novel natural product exhibiting cytotoxic activity through microtubule stabilization.
• CS is the sole known microtubule-stabilizing agent (MSA) that forms a covalent bond with tubulin.
• CS displays rapid binding kinetics, similar to other MSAs.

Purpose of the Study:

• To investigate the binding site and mechanism of action of cyclostreptin (CS) on tubulin.
• To challenge the proposed low-affinity binding site for CS and offer an alternative mechanistic explanation.
• To elucidate the structural basis for CS-induced microtubule stabilization.

Main Methods:

• Peptide digestion and mass spectrometry analysis to identify CS-labeled amino acids.
• Molecular dynamics simulations to study tubulin dimer and microtubule pore interactions.
• Structural analysis of the microtubule pore and its role in ligand accessibility.

Main Results:

• CS covalently binds to Asn228 (near the taxane-binding site) and Thr220 (in the microtubule pore).
• The microtubule pore, not a low-affinity site, presents a kinetic barrier to CS binding at the taxane site.
• CS binding at Thr220 blocks access to the microtubule lumen.
• Simulations indicate MSAs targeting the taxane site stabilize microtubules via M-loop conformational changes.

Conclusions:

• The microtubule pore serves as a kinetic barrier, obviating the need for a distinct low-affinity CS binding site.
• CS binding at Thr220 within the pore effectively blocks the entry to the microtubule lumen.
• A common mechanism of microtubule stabilization for taxane-site MSAs involves M-loop conformational changes.