Functional role of arginine-11 in the N-terminal helix of skeletal troponin C: combined mutagenesis and molecular dynamics investigation.
The two main structural differences between calmodulin (CaM) and skeletal troponin C (sTnC) are the absence in CaM of (i) the short N-terminal helix in TnC and (ii) the triplet KGK (residues 91-93; numbering according to chicken sTnC). It was recently shown that deletion of both structural groups from sTnC imparted to the resulting construct the CaM-like ability to activate phosphodiesterase (PDE) and to regulate force development in smooth muscle. To continue probing of the structural basis of the differential behavior of sTnC and CaM, residue Arg-11 in rabbit sTnC was mutated to Ala because the interactions of Arg-11 with distal residues in the N-terminal domain seem to link the N-terminal helix to the rest of the structure. The mutant exhibits CaM-like function in its ability to activate PDE (about 50% of CaM at 5 microM concentration). If, in addition, the KGK triplet is also deleted, PDE activation increases to about 80%. Both constructs retain their TnC function to nearly 100%. To explore the mechanistic basis of this remarkable observation, computational simulations of the molecular dynamics (MD) were carried out for both wild-type 4Ca2+.sTnC and the 4Ca2+.R11A mutant, and the results were compared to those from earlier simulations of 4Ca2+.CaM. Two types of structural changes observed from such simulations of the molecular dynamics of CaM had been considered to have a functional role: (i) a compaction to a more globular form and (ii) a reorientation of the Ca-binding domains around the central tether helix.(ABSTRACT TRUNCATED AT 250 WORDS)
Gulati
J
J
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Akella
A B
AB
Su
H
H
Mehler
E L
EL
Weinstein
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D003196:Computer Graphics; D003198:Computer Simulation; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014336:Troponin / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D019209:Troponin C
D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D001120:Arginine