Molecular Characterisation of Titin N2A and Its Binding of CARP Reveals a Titin/Actin Cross-linking Mechanism |
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Affiliation: | 1. Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6–3, Aramaki, Aoba-ku, Sendai 980–8578, Japan;2. Department of Pharmacology, Yamagata University School of Medicine, Iida-Nishi 2-2-2, Yamagata 990–9585, Japan;3. Department of Physiology and Pharmacology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USA;4. The Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USA;5. Department of Urology, Yamagata University School of Medicine, Iida-Nishi 2-2-2, Yamagata 990-9585, Japan;1. Department of Biology, University of Konstanz, 78457 Konstanz, Germany;2. Institute of Biophysics and Physical Biochemistry, University of Regensburg, 93040 Regensburg, Germany;3. Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK;1. Department of Chemistry and Biochemistry, Northern Arizona University, Flagstaff, AZ, United States;2. Department of Biology, Northern Arizona University, Flagstaff, AZ, United States;3. Center for Bioengineering Innovation, Northern Arizona University, Flagstaff, AZ, United States;4. Department of Chemistry, University of Massachusetts Lowell, Lowell, MA, United States |
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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. |
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Keywords: | nuclear magnetic resonance hydrogen–deuterium exchange mass spectrometry muscle stress response actin cytoskeleton sarcomere mechanics CARP" },{" #name" :" keyword" ," $" :{" id" :" k0035" }," $$" :[{" #name" :" text" ," _" :" cardiac ankyrin repeat protein DAPI" },{" #name" :" keyword" ," $" :{" id" :" k0045" }," $$" :[{" #name" :" text" ," _" :" 4′,6-diamidino-2-phenylindole EDTA" },{" #name" :" keyword" ," $" :{" id" :" k0055" }," $$" :[{" #name" :" text" ," _" :" Ethylenediaminetetraacetic acid GFP" },{" #name" :" keyword" ," $" :{" id" :" k0065" }," $$" :[{" #name" :" text" ," _" :" green fluorescent protein HDX-MS" },{" #name" :" keyword" ," $" :{" id" :" k0075" }," $$" :[{" #name" :" text" ," _" :" Hydrogen/Deuterium exchange mass spectrometry HSQC" },{" #name" :" keyword" ," $" :{" id" :" k0085" }," $$" :[{" #name" :" text" ," _" :" heteronuclear single quantum correlation Ig" },{" #name" :" keyword" ," $" :{" id" :" k0095" }," $$" :[{" #name" :" text" ," _" :" immunoglobulin LC-MS" },{" #name" :" keyword" ," $" :{" id" :" k0105" }," $$" :[{" #name" :" text" ," _" :" Liquid chromatography–mass spectrometry MARP, muscle ankyrin repeat protein MLP" },{" #name" :" keyword" ," $" :{" id" :" k0115" }," $$" :[{" #name" :" text" ," _" :" muscle Lim protein MS" },{" #name" :" keyword" ," $" :{" id" :" k0125" }," $$" :[{" #name" :" text" ," _" :" Mass spectrometry MyBP-C3" },{" #name" :" keyword" ," $" :{" id" :" k0135" }," $$" :[{" #name" :" text" ," _" :" Myosin Binding Protein C3 NMR" },{" #name" :" keyword" ," $" :{" id" :" k0145" }," $$" :[{" #name" :" text" ," _" :" Nuclear magnetic resonance NOE" },{" #name" :" keyword" ," $" :{" id" :" k0155" }," $$" :[{" #name" :" text" ," _" :" nuclear Overhauser effect PCR" },{" #name" :" keyword" ," $" :{" id" :" k0165" }," $$" :[{" #name" :" text" ," _" :" polymerase chain reaction RCI" },{" #name" :" keyword" ," $" :{" id" :" k0175" }," $$" :[{" #name" :" text" ," _" :" random coil index RFDU" },{" #name" :" keyword" ," $" :{" id" :" k0185" }," $$" :[{" #name" :" text" ," _" :" relative fractional deuterium uptake RMSD" },{" #name" :" keyword" ," $" :{" id" :" k0195" }," $$" :[{" #name" :" text" ," _" :" root-mean-square deviation TCEP" },{" #name" :" keyword" ," $" :{" id" :" k0205" }," $$" :[{" #name" :" text" ," _" :" Tris(2-carboxyethyl)phosphine hydrochloride UN2A" },{" #name" :" keyword" ," $" :{" id" :" k0215" }," $$" :[{" #name" :" text" ," _" :" unique N2A sequence |
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