Structural characterization of the proteic complex Calsarcin 1: Calcineurin A

Author(s)

Carlos Roberto Koscky Paier

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2014-08-15

Examining board members

Kleber Gomes Franchini; Adriana Franco Paes Leme; Jörg Kobarg; Daniela Barretto Barbosa Trivella; Juliana Ferreira de Oliveira

• Advisor: Kleber Gomes Franchini

Abstract

Signaling by the calcium-dependent phosphatase calcineurin (Cn) plays key roles in regulating cardiac development, hypertrophy, and pathological remodeling. Cn binds to and is negatively regulated by calsarcins (CS), a family of muscle-specific proteins. However, the molecular mechanisms involved in the inhibition of Cn by CS remain unclear. Understanding the architecture and structure of Cn-CS complex is critical to unravel the regulation of Cn by CS. Here we combined biochemical assays, chemical crosslinking coupled to mass spectrometry experiments (MS/MS), mutational analysis and a modeling strategy for structural characterization of CnA-CS1 assembly. The MS/MS data obtained from the cross-linked peptides of both proteins were used to guide an in silico docking of their polypeptide models. The protein complex models with the smallest estimated binding energy were clustered according to structural similarity and submitted to molecular dynamics simulation. The interacting surface of CnA was mapped in a pocket between the 1st and 3rd ?-helixes and surrounding loops, while the corresponding surface of CS1 was mapped to the carboxyterminal loops within the Leu179-Phe185, Phe195-Ser199 and Thr250-Leu264 regions. Notably, the region of CnA that interacts with CS1 was found to be located in close proximity, but not coincident, to the ?-sheet 14, the main binding site for the PVIVIT sequence of NFAT. Experiments performed with several CnA (FLAG-CnA) and CS1 (myc-CS1) mutants were used to validate the structural model of the CnA-CS1 assembly. The Lys40 (CnA) and Glu254 (CS1) residues were identified as critical for the complex stability. The model that emerges from this study supports the notion that CS1 interacts with an allosteric site to inhibit the activity of CnA (AU)

• FAPESP's process: 11/01356-8 - Functional and Structural Analysis of the Cardiac Proteins Associated to the Z Disc, Calsarcin and Enigma
• Grantee: Carlos Roberto Koscky Paier
• Support Opportunities: Scholarships in Brazil - Doctorate