Dynamic-Structure Redesign of Calmodulin Reveals Mechanistic Constraints on Ryr2 Regulation

Area of Science:

• Biochemistry
• Structural Biology
• Computational Biology

Background:

• Calmodulin (CaM) is a vital calcium (Ca2+) sensor regulating numerous cellular processes.
• CaM's evolutionary conservation and flexibility make it challenging to redesign rationally.
• Understanding CaM's role in Ca2+ signaling is critical for disease intervention.

Purpose of the Study:

• To investigate if incorporating conformational dynamics into computational protein design can enable functional reengineering of CaM.
• To test a dynamic-structure redesign strategy for CaM using the Ryanodine receptor 2 (RyR2) as a model.
• To determine if enhanced binding affinity alone is sufficient for improved CaM function.

Main Methods:

• Static structure-based computational protein design to increase CaM-RyR2 affinity.
• Molecular dynamics simulations to guide a dynamic-structure redesign strategy.
• In vitro binding assays and ex vivo functional assays in cardiomyocytes.

Main Results:

• Static redesign increased CaM-RyR2 binding but distorted RyR2 peptide and worsened Ca2+ leak.
• Dynamic-structure redesign preserved CaM's conformational integrity and enhanced RyR2 binding.
• The dynamic CaM variant reduced pathological Ca2+ leak in a disease model.

Conclusions:

• Successful CaM redesign necessitates preserving its conformational dynamics, not just increasing binding affinity.
• Integrating conformational dynamics into protein design enables predictive engineering of flexible protein-protein interactions.
• This approach holds promise for developing therapeutics targeting Ca2+ signaling pathways and related diseases.