Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism

J Mol Biol. 2021 Apr 30;433(9):166901. doi: 10.1016/j.jmb.2021.166901. Epub 2021 Feb 27.

Abstract

Striated muscle responds to mechanical overload by rapidly up-regulating the expression of the cardiac ankyrin repeat protein, CARP, which then targets the sarcomere by binding to titin N2A in the I-band region. To date, the role of this interaction in the stress response of muscle remains poorly understood. Here, we characterise the molecular structure of the CARP-receptor site in titin (UN2A) and its binding of CARP. We find that titin UN2A contains a central three-helix bundle fold (ca 45 residues in length) that is joined to N- and C-terminal flanking immunoglobulin domains by long, flexible linkers with partial helical content. CARP binds titin by engaging an α-hairpin in the three-helix fold of UN2A, the C-terminal linker sequence, and the BC loop in Ig81, which jointly form a broad binding interface. Mutagenesis showed that the CARP/N2A association withstands sequence variations in titin N2A and we use this information to evaluate 85 human single nucleotide variants. In addition, actin co-sedimentation, co-transfection in C2C12 cells, proteomics on heart lysates, and the mechanical response of CARP-soaked myofibrils imply that CARP induces the cross-linking of titin and actin myofilaments, thereby increasing myofibril stiffness. We conclude that CARP acts as a regulator of force output in the sarcomere that preserves muscle mechanical performance upon overload stress.

Keywords: actin cytoskeleton; hydrogen–deuterium exchange mass spectrometry; muscle stress response; nuclear magnetic resonance; sarcomere mechanics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / chemistry*
  • Actins / metabolism*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Connectin / chemistry*
  • Connectin / metabolism*
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / metabolism
  • Male
  • Mice
  • Muscle Proteins / chemistry
  • Muscle Proteins / genetics
  • Muscle Proteins / metabolism*
  • Mutation
  • Myofibrils / chemistry
  • Myofibrils / metabolism
  • Nuclear Magnetic Resonance, Biomolecular
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Pliability
  • Protein Binding
  • Rabbits
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism*
  • Sarcomeres / chemistry
  • Sarcomeres / metabolism

Substances

  • ANKRD1 protein, human
  • Actins
  • Connectin
  • Cross-Linking Reagents
  • Muscle Proteins
  • Nuclear Proteins
  • Repressor Proteins