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MRE222x103[degxcm2xdmol-1]
M7aSAH
ccM2
Insights into the molecular mechanisms of myosin 7a regulation
Glenn Carrington1, Robert Welch2, Marcin Wolny1, Sarah Harris2, Daniel Read3, Oliver Harlen3, Michelle Peckham1
bs10g3c@leeds.ac.uk,s.a.harris@leeds.ac.uk,d.j.read@leeds.ac.uk,o.j.harlen@leeds.ac.uk,M.peckham@leeds.ac.uk
1 Faculty of Biological Sciences, University of Leeds, LS2 9JT 2 School of Physics and Astronomy, University of Leeds, LS2 9JT 3 School of Mathematics, University of Leeds, LS2 9JT
Introduction
• Myosin 7a is an unconventional molecular motor that is able to
regulate its activity through a head-tail interaction[1].
• This process spans many length and timescales, with large-scale
protein dynamics, as well as atomistic contributions leading to the
formation of the compact structure.
• Understanding how this process occurs will allow further insight
into the action of Myosin 7a.
Objectives
Regulated Extended
Negative-stain electron microscopy images of myosin 7a in its regulated and
extended form. The tip of the C-terminal FERM domain interacts with the motor
domain. Scale bar 10nm.
This project aims to simulate the formation of the regulated
structure of myosin 7a using a number of simulation techniques.
So far, we have investigated:
• How flexible do the subdomains of the lever need to be for the
molecule to fold up.
Crystal
structures: Homology
model: Structure
prediction:
As there is very little structural information about myosin 7a, an all
atom model was constructed using available crystal structure,
homology modelling and structural prediction programs. Careful
construction of the model was necessary as this model would form
the basis of oursimulation techniques.
Construction of all-atom model
To overcome the computational cost of the all-atom molecular
dynamics (MD) approach, we have developed a novel coarse-grained
(CG) simulation approach, named Fluctuating Finite Element Analysis[2]
(FFEA), which:
• Models globular proteins as continuum
• Subject to thermalfluctuations
• No need for atomic structure
Solve the Cauchy momentum equation:
ρ!"
!#
=
∇·( σv + σe ) + f + ∇·π
FFEA model
Where σv
is viscous stress, σe
is elastic stress, f is external forces, and
π is thermal noise.
Iteratively within the volume of each protein in order to compute MD
trajectories. We do so through Finite Element Analysis and therefore
molecules need to be discretised, in our case using a tetrahedral mesh:
Coarse-­‐graining
gradient
How flexible does the neck need to be to adopt the compact form?
The bending stiffness of the molecule is defined through the Young’s
modulus [E]. Homogeneous parameters obtained from the dynein stalk
region (B)[2] and (C) predictions of the bending stiffness of the lever
(≈1500pN·nm2) and the SAH (154pN·nm2)[3] were used to parameterise
models. (A) shows the flexibility required forinteraction.
CA B
Myosin 7a is monomeric – the SAH domain does not dimerise
i)
The myosin 7a SAH melts non-cooperatively as expected of a
typical SAH domain (i). Gel filtration, which separates proteins on
their MW and shape, shows that myosin 7a SAH is an elongated
monomeric structure(ii).
Myosin
7a
end-­‐to-­‐end
distance
As the neck becomes more flexible, the head and tail are able to get
muchcloser together, required for regulation. In (A)the motor andtail are
sufficiently close that interaction could occur. However, these values are
extremely low, withstructured proteins usually having a Young’s modulus
on order of hundreds of Mpa[4]. Our simulations suggest that the flexibility
of the SAH domain must be highto allow folding.
Scan
Me
Conclusions / Future prospects
• As a defined structure, the neck region needs to be extremely flexible
in order for the molecule to form the regulatedstate.
• In our model, all IQ motifs are occupied by calmodulin, although in
reality this may not be thecase. Experiments are underway to assess
which light chains myosin 7a binds underdifferent conditions.
References
[1] Y. Yang et al., P.N.A.S. 106 11(2009)
[2] R. C. Oliver et al.,J.Comput. Phys.239,147 (2013).
[3] S. Sivaramakrishnanet al.,ProcNatl Acad Sci 105, 36(2008).
[4] M. Guthold et al. CellBiochemBiophys. 49,3 (2007).
2215
Motor Lever SAH Tail
6 8 10 12
0.0
0.5
1.0
Elution volume [ml]
NormalisedAU
RNase A mass: 13.7 kDa
M7aSAH mass: 9.9 kDa
11 heptads coiled-coil fragment
dimer mass: 18.3 kDa
ii)
6 8 10 12
0.0
0.5
1.0
Elution volume [ml]
NormalisedAU
RNase A mass: 13.7 kDa
M7aSAH mass: 9.9 kDa
11 heptads coiled-coil fragment
dimer mass: 18.3 kDa
SAH
stiffness [E] 33.75MPa 270MPa 382MPa
Lever
stiffness [E] 67.5MPa 270MPa 827MPa
Scan
Me Scan
Me
For regions without crystal structures or that could not be homology modelled,
structural prediction programs were used. The region connecting the SAH domain
and the tail (936-1016) was predicted to be mostly unstructured.