Molecular Basis of Calcium-Sensitizing and Desensitizing
Mutations of the Human Cardiac Troponin C Regulatory
Domain: A Multi-Scale Simulation Study
Peter Michael Kekenes-Huskey*, Steffen Lindert, James Andrew McCammon
Department of Pharmacology, Center for Theoretical Biological Physics, National Computational Biomedical Resource and Howard Hughes Medical Institute, University of
California San Diego, La Jolla, California, United States of America
Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic Ca2zand subsequent
recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown
to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of
engineered TnC variants that increase or decrease Ca2zsensitivity, in order to understand the structural basis of their
impact on TnC function. We will use the distinction for mutants that are associated with increased Ca2zaffinity and for
those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the
physiologically-active site II Ca2zbinding site in the Ca2z-free (apo) state closely resembled the Ca2z-bound (holo) state.
In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo
conformation. We hypothesize that these phenomena contribute to the increased association rate, kon, for the GOF mutants
relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the
GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to
Ca2zaffinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC
provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs
that target TnC.
Citation: Kekenes-Huskey PM, Lindert S, McCammon JA (2012) Molecular Basis of Calcium-Sensitizing and Desensitizing Mutations of the Human Cardiac
Troponin C Regulatory Domain: A Multi-Scale Simulation Study. PLoS Comput Biol 8(11): e1002777. doi:10.1371/journal.pcbi.1002777
Editor: Roland L. Dunbrack, Fox Chase Cancer Center, United States of America
Received April 3, 2012; Accepted September 28, 2012; Published November 29, 2012
Copyright: ? 2012 Kekenes-Huskey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Institutes of Health (www.nih.gov), the National Science Foundation (www.nsf.gov), the Howard Hughes
Medical Institute (www.hhmi.org), the National Biomedical Computation Resource (nbcr.net), and the NSF Super- computer Centers (www.xsede.org). Molecular
dynamics computations were performed on the DAVinCI cluster acquired with funds from NSF grant OCI-0959097 (www.rcsg.rice.edu/davinci/). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Sarcomeres contract owing to the translocation of the thick
filament, comprised of myosin, along actin chains constituting the
thin filament (TF). Contraction is initiated and regulated by
Troponin proteins tethered to actin, including Troponin C (TnC),
Troponin I (TnI) and Troponin T (TnT), as well as Tropomyosin
(Tm). Specifically, Ca2zbinds to TnC, thereby unveiling a
hydrophobic region necessary for binding the TnI switch peptide.
Liberation of the TnI regulatory unit from the TF initiates a shift
in Tm , thus enabling the weak binding of myosin to actin.
Subsequent conversion of Tm to the unblocked state permits a
cycle of strong myosin binding and propagation along the TF
A number of human cardiac diseases including hypertrophic
cardiomyopathy (HCM) , restrictive cardiomyopathy (RCM)
 and dilated cardiomyopathy (DCM)  have been attributed
to mutations in thin filament, thick filament and associated
proteins of the sarcomere. RCM and HCM mutations have been
shown to increase Ca2zsensitivity of force generation as measured
by pCa50, whereas DCM mutations reduce this trend. A large
number of mutations leading to HCM, RCM, and DCM
phenotypes have been collectively identified  but only one
LOF, DCM-associated mutation has been found in TnC (D75Y
). The prominent role of TnC in force development has thus
attracted therapeutic strategies to tune its Ca2zand TnI affinity
including drug-design  and protein engineering approaches [8–
10]. In particular, mutation studies of full-length TnC have
revealed engineered variants that shift the Ca2zequilibrium
constant, Keq(or pCa50), leading to altered force development akin
to GOF and LOF . For instance, V44Q and L48Q mutations
investigated by [8,10,11] have been reported to exhibit GOF-like
phenotypes in skinned cardiac fibers, with pCa50values of 6.29
and 6.13 (in isolated F27W TnC), respectively, relative to the wild-
type value of 5.48 . Furthermore, Tikunova et al.  reported
for several GOF mutations including V44Q and L48Q that faster
Ca2zassociation rates (4.4 to 5.2-fold) contributed more to the
increased Keqrather than slowed dissociation (approximately 2.8-
fold). In comparison, the E40A and V79Q mutations examined by
 present LOF-like alterations in force generation with pCa50
values of 5.16 and 5.30, respectively. While these studies have
implicated Ca2zbinding as the primary factor in reshaping
PLOS Computational Biology | www.ploscompbiol.org1 November 2012 | Volume 8 | Issue 11 | e1002777
contractile activity, the structural and dynamical basis of the
mutations’ effect on TnC is largely unknown.
Structure determination via X-ray crystallography  and
NMR [13–15] has yielded important insight into the molecular
basis of TnC function. These studies indicate that TnC consists of
two domains: the C-terminal domain is affixed to the thin
filament, while the N-terminal regulatory domain is responsible for
binding Ca2zat physiological concentrations, and TnI. The TnC
N-terminal domain consists of five helices, HN (4–8), HA (14–24),
HB (38–48), HC (54–64) and HD(73–85), two beta sheets b1 (36–
37), b2 (71–72) (Fig. 1). Loops LAB(25–35), LCD(65–70) of sites I
and II form EF hands (helix-loop-helix) that selectively bind Ca2z,
although in cardiac TnC, only site II is physiologically active.
Within the EF hand, several acidic residues (D65, D67, and E76)
coordinate Ca2z, along with S69 and T71; we collectively refer to
these amino acids as chelation residues.
Prior experimental and theoretical work have leveraged these
structural data to probe rapid, nanosecond timescale conforma-
tional dynamics that are correlated with Ca2zbinding [14,16].
Lim and coworkers  characterized a TnC mutant (D75Y)
isolated from a patient with DCM and demonstrated its decreased
Ca2zbinding capacity and disruption of normal structural
dynamics. Varughese and Li further investigated via MD changes
in the structural dynamics of cardiac troponin, including TnC,
upon binding bepridil, a known inotropic agent . Lindert and
coworkers  combined long time-scale MD simulations and BD
simulations to understand the dynamics of wild-type TnC in its
Ca2z-free, Ca2z-bound, and TnI -bound states, as well as V44Q
. Recently, a combined experimental and theoretical approach
examined Ca2zbinding and the structural stability of a GOF
mutant L48Q .
We seek to extend these studies by 1) comparing LOF and GOF
mutants to better contrast differences in apparent Ca2zsensitivity
and 2) explore longer simulation times comparable to dynamics
captured by NMR order parameters. By a combination of
molecular and Brownian dynamics, our approach identifies
structural and dynamic factors impacting Ca2zbinding in light
of GOF and LOF mutations. The outcome of this study provides
greater insight into the mechanisms of structure/function
relationships for N-terminal cardiac TnC that are important to
Mutations slightly disrupt wild-type TnC structure
Overall TnC structure.
and LOF- associated mutations on the structure of the N-terminal
regulatory domain of TnC and subsequent impact on Ca2z
affinity, we performed in silico mutations of the Ca2z-bound (holo)
and Ca2z- free (apo) wild-type NMR structures (1AP4 and
1SPY). The mutations included the GOF-like mutants V44Q and
L48Q, as well as the LOF-like mutants V79Q and E40A. These
mutants induced significant changes in the polar character of wild-
type TnC, with V44Q, L48Q, and V79Q representing apolar to
polar alterations and E40A, a charged to neutral substitution. It is
plausible that these mutations might disrupt the hydrophobic
packing of the wild-type TnC structure . Our simulations
indicated that the mutations preserved the overall arrangement of
helices as evidenced by the superimposed structures in Fig. 2 after
equilibration, with relatively minor Ca RMSD differences with
respect to wild-type (RMSDƒ3:0A) (Fig. S2a). While the Ca
RMSD does not strongly distinguish between GOF and LOF
mutants, differences were noted at LCD, the physiologically active
Ca2zbinding domain, with increasing deviations noted for V44Q,
E40A, V79Q, and L48Q (RMSD from 1.0 to 3.5 A˚, (Fig. S2c).
Differences between structures were comparatively less apparent
for all other helices and loops. An exception to this trend was
noted for V44Q, which displayed significant RMSD changes for
HB, HDand LAB. In the subsequent results and discussion, we will
argue that these deviations likely corresponded to low-frequency,
collective motions involved in TnI recognition.
Local conformation and electrostatic potential.
such similar backbone configurations noted amongst the mutants,
we examined whether changes in electrostatic potential, particu-
larly near the mutation sites, could account for altered Ca2z
association rates. In the wild-type structure, the native residues at
the apolar mutation sites in wild-type TnC were well-buried, while
To understand the impact of GOF-
Figure 1. Structure of wild-type cardiac Troponin C. The
physiologically inactive Ca2zbinding site I contains LAB (yellow),
while the active site II region contains LCD(tan). The locations of the
mutants in this study are marked as colored bands, including E40
(black) V44 (red), L48 (green) and V79 (blue).
Muscle cells contract using a network of thread-like
protein assemblies called myofilaments. Contraction is
preceded by a signal that causes calcium to rush into the
cell cytosol, where it can freely diffuse to and bind the
myofilament proteins. Troponin C, a calcium sensor
located on the thin filament, initiates and regulates the
cascade of changes resulting in the generation of force by
the thin and thick filaments comprising the myofilament
lattice. In heart tissue, pathological conditions known as
dilated and hypertrophic cardiomyopathies (DCM and
HCM, respectively) are in part associated with abnormal-
ities in the ability of the myofilaments to generate force at
normal calcium concentrations. Manipulation of Troponin
C calcium-binding through protein engineering and
pharmaceutical intervention has thus attracted consider-
able attention as a therapeutic strategy for ameliorating
these cardiac defects. In this study, we uncover a
molecular basis of altered calcium handling for several
engineered Troponin C variants, which provides further
insight into tuning its control of myofilament contraction.
Cardiac Troponin C Mutations
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