Functional Role of Arginine-11 in the N-Terminal Helix of Skeletal Troponin C: Combined Mutagenesis and Molecular Dynamics Investigation

Abstract

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)

Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 Å resolution

“…Loss of Arg11 or the N helix in general would destabilize loop II and cause the observed decrease in calcium af®nity and calcium-regulated activity. Our observation disputes earlier molecular dynamic simulations on a modelled 4-Ca 2 -TnC, which suggested that upon calcium uptake, Arg11 and Glu76 would signi®cantly break apart such that their interaction was via a network of water molecules rather than via a direct ion pair (Gulati et al, 1995).…”

Mutations in the N- and D-Helices of the N-Domain of Troponin C Affect the C-Domain and Regulatory Function

“…The prediction that the loss of Arg 11 (or the salt bridge) would destabilize loop 2 and decrease Ca 2ϩ affinity was not borne out by our assays, although deletion of the entire N-helix does reduce the Ca 2ϩ affinity of the N-domain (Chandra et al, 1994;Smith et al, 1994). Our results are in agreement with Gulati et al (1995) who reported that an R11A mutant could restore full tension regulation in TnC-depleted fibers.…”

A new method for modeling large-scale rearrangements of protein domains

“…Most often, the difficulty of escaping from the close vicinity of a starting conformation was found to be formidable. Broader sampling of the conformational space could be achieved if the covalent or intradomain structure of a protein was preserved by a series of restraints, [26][27][28] and/or atomic masses were artificially increased. 29 Among other methods that may be applicable to large-scale domain rearrangement modeling, are random and friction forces molecular dynamics, 30 internal coordinate molecular dynamics, [31][32][33][34] stochastic rigid body, and brownian dynamics with simplified residue representation, [35][36] Monte Carlo calculations in space of scaled collective internal variables, 37 and ''essential dynamics.''…”