Protein flexibility is key to cisplatin crosslinking in calmodulin

Area of Science:

• Proteomics
• Structural Biology
• Computational Biology

Background:

• Chemical crosslinking coupled with mass spectrometry is vital for protein structure and interaction studies.
• Cisplatin has been identified as a crosslinker for methionine residues in apo-calmodulin (apo-CaM), but experimental distances conflict with structural data.

Purpose of the Study:

• To investigate the mechanism of cisplatin crosslinking in calmodulin (CaM) despite apparent distance constraints.
• To explore the role of protein flexibility in facilitating crosslinking events.
• To assess the impact of cisplatin-induced crosslinking on CaM's functional interactions.

Main Methods:

• Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for identifying crosslinked residues.
• Novel computational flexibility analysis to assess protein dynamics.
• In vitro binding assays comparing modified and unmodified CaM with melittin.

Main Results:

• Computational flexibility analysis revealed that CaM's dynamic motions allow methionine side chains to approach for cisplatin crosslinking in both apo-CaM and Ca₄-CaM.
• FTICR MS data confirmed cisplatin-mediated crosslinking in Ca₄-CaM.
• Cisplatin crosslinking was shown to inhibit CaM's binding to its target proteins, melittin.

Conclusions:

• Protein flexibility analysis is a valuable tool for predicting crosslinking sites and aiding mass spectrometry data interpretation.
• Cisplatin crosslinking of CaM interferes with its biological function by blocking target protein binding.
• These findings offer insights into platinum-based anticancer drug resistance mechanisms.