DEER experiments reveal fundamental differences between calmodulin complexes with IQ and MARCKS peptides in solution

Area of Science:

• Structural Biology
• Biochemistry
• Calcium Signaling

Background:

• Calmodulin (CaM) is a crucial calcium-binding protein regulating numerous cellular functions.
• CaM undergoes significant conformational changes upon binding to its target partners, influencing protein activity.
• Understanding CaM's solution binding modes is essential for elucidating its regulatory mechanisms.

Purpose of the Study:

• To compare the solution binding modes of CaM with target peptides MARCKS and IQ.
• To investigate the conformational changes of CaM upon binding to these peptides in solution.
• To determine the relative orientation of CaM domains during peptide binding.

Main Methods:

• Utilized double electron-electron resonance (DEER) distance measurements and paramagnetic NMR.
• Employed nitroxide and Gd(III) spin labels, including Gd(III) substitution for Ca²⁺ in a CaM mutant.
• Integrated elastic network modeling with experimental distance restraints.

Main Results:

• MARCKS binding to holo-CaM in solution did not result in a closed conformation or a unique domain orientation, differing from crystal structures.
• IQ binding to holo-CaM induced a closed conformation in solution.
• A solution structure model for holo-CaM/IQ was derived using 12 DEER distance restraints and elastic network modeling, showing good agreement with crystal structures.

Conclusions:

• CaM exhibits distinct solution binding modes with different target peptides.
• The crystal structure may not fully represent the dynamic solution behavior of CaM-peptide complexes.
• DEER and computational modeling provide valuable insights into CaM's conformational dynamics in solution.