Calcium Causes a Conformational Change in Lamin A Tail
Domain that Promotes Farnesyl-Mediated Membrane
Association
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Citation
Kalinowski, Agnieszka, Zhao Qin, Kelli Coffey, Ravi Kodali,
Markus J. Buehler, Mathias Losche, and Kris Noel Dahl.
“Calcium Causes a Conformational Change in Lamin A Tail
Domain That Promotes Farnesyl-Mediated Membrane
Association.” Biophysical Journal 104, no. 10 (May 2013):
2246–2253. © 2013 Biophysical Society
As Published
http://dx.doi.org/10.1016/j.bpj.2013.04.016
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Elsevier
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Thu May 26 02:01:03 EDT 2016
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2246
Biophysical Journal
Volume 104
May 2013
2246–2253
Calcium Causes a Conformational Change in Lamin A Tail Domain that
Promotes Farnesyl-Mediated Membrane Association
Agnieszka Kalinowski,†6 Zhao Qin,‡6 Kelli Coffey,†§ Ravi Kodali,{ Markus J. Buehler,‡* Mathias Lösche,†k***
and Kris Noel Dahl†§*
†
Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania; ‡Civil and Environmental Engineering, Massachusetts
Institute of Technology, Cambridge, Massachusetts; §Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania;
{
Department of Structural Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; kDepartment of Physics, Carnegie Mellon University,
Pittsburgh, Pennsylvania; and **Center for Neutron Research, NIST, Gaithersburg, Maryland
ABSTRACT Lamin proteins contribute to nuclear structure and function, primarily at the inner nuclear membrane. The posttranslational processing pathway of lamin A includes farnesylation of the C-terminus, likely to increase membrane association,
and subsequent proteolytic cleavage of the C-terminus. Hutchinson Gilford progeria syndrome is a premature aging disorder
wherein a mutant version of lamin A, D50 lamin A, retains its farnesylation. We report here that membrane association of farnesylated D50 lamin A tail domains requires calcium. Experimental evidence and molecular dynamics simulations collectively
suggest that the farnesyl group is sequestered within a hydrophobic region in the tail domain in the absence of calcium. Calcium
binds to the tail domain with an affinity KD z 250 mM where it alters the structure of the Ig-fold and increases the solvent accessibility of the C-terminus. In 2 mM CaCl2, the affinity of the farnesylated protein to a synthetic membrane is KD z 2 mM, as
measured with surface plasmon resonance, but showed a combination of aggregation and binding. Membrane binding in the
absence of calcium could not be detected. We suggest that a conformational change induced in D50 lamin A with divalent cations plays a regulatory role in the posttranslational processing of lamin A, which may be important in disease pathogenesis.
INTRODUCTION
A-type lamins, including lamin A (LA), contribute to a
variety of functions that include DNA transcription, heterochromatin regulation, cell senescence, DNA damage repair,
and the sequestration of transcription factors (recent reviews
(1,2)). These type V intermediate filament proteins are also
the primary structural elements of the nucleoskeleton (3).
Lamins are structurally related to other intermediate filament proteins (4), but the C-terminal tail domain (TD) of
LA is much larger than that of other intermediate filaments
and undergoes significant posttranslational processing.
LMNA codes for the precursor prelamin A (preLA), which
is transported into the nucleus where the C-terminal CaaX
domain (specifically, Cys-Ser-Ile-Met) is farnesylated and
carboxymethylated (5). The modified preLA is then cleaved
by the enteroprotease ZMPSTE-1 at Y646 to produce
mature LA. Dysfunctions of this process including loss of
ZMPSTE-1 or mutations including the Y646 cleavage site
are responsible for devastating human diseases. In the premature aging syndrome Hutchinson Gilford progeria syndrome (HGPS), a C1824T mutation of LMNA causes the
activation of a cryptic splice site and a loss of exon 11 in
the TD (6). The loss of the 50 amino acid segment, which
contains the ZMPSTE-1 cleavage region, causes retention
of the farnesylation, which in turn results in changes in ther-
Submitted November 29, 2012, and accepted for publication April 8, 2013.
6
Agnieszka Kalinowski and Zhao Qin contributed equally to this work.
*Correspondence: mbuehler@mit.edu or quench@cmu.edu or krisdahl@
cmu.edu
modynamic stability of the mutant protein, D50LA or
progerin (7). At a much lower frequency than in HGPS, activation of the cryptic splice site that produces D50LA occurs
sporadically in normal cells, and this defect accumulates
with aging (8).
The biochemistry of LA TD processing has been determined, but the biological benefits of the complex processing
pathway and the regulation of its individual steps are unclear
(9). Although farnesyl transferases have been found in both
the endoplasmic reticulum and the nucleus, farnesylation
of the protein likely occurs in the nucleus (5). The farnesyl
modification is suggested to increase the association of
preLA with the inner nuclear membrane (10,11). Similarly,
the pathologies associated with the expression of D50LA in
HGPS result, in part, from the farnesylation causing overaccumulation of structural proteins at the inner nuclear
membrane (12,13). Indeed, mislocalization of LA and LAassociated proteins at the inner nuclear membrane results
in altered DNA repair, transcription, and signaling (14), as
well as changes in protein-protein interactions (15). On the
other hand, a single farnesylation is insufficient to confer
permanent membrane association of proteins in other systems (16–18). Therefore, it is unclear if the presence of the
farnesyl is sufficient for the cellular pathological phenotype
or if additional factors, including altered protein-protein interactions (15) or altered protein packing (7,19) are involved.
For the full-length lamin protein, the rod domain functions in dimer formation and the head and tail domains associate with the rod to form filaments; full-length filaments
form aggregates and paracrystals in vitro (1–4). The TD is
Editor: David Odde.
Ó 2013 by the Biophysical Society
0006-3495/13/05/2246/8 $2.00
http://dx.doi.org/10.1016/j.bpj.2013.04.016
Farnesylated Lamin A Tail Domain
responsible for association with other proteins and membrane localization. For these reasons, we limited our study
to the TD to isolate the interaction between the protein
and the membrane (2,10). Here, we quantify protein-membrane binding of farnesylated D50LA (fn-D50LA) by investigating purified LA TDs in solution and on model lipid
bilayer membranes. We find that the binding is weak
(KD ~2 mM) and that free Ca2þ is required for binding.
The conformational response of the protein to Ca2þ is
tracked by monitoring its intrinsic fluorescence. Molecular
simulations suggest that the Ca2þ-induced conformational
change opens the Ig-fold, increases the flexibility of the
terminal CaaX motif, and exposes the farnesyl group, which
is sequestered within the TD in the absence of the ion.
2247
has four tryptophan residues all of which are located on or near the Ig-fold.
The peak emission intensity is at 342 nm, after excitation at 298 nm to minimize excitation of other aromatic residues. A peak emission at 342 nm
indicates that the tryptophan residues are neither in a completely hydrophobic environment, nor are they fully solvent exposed (20,21).
The peak emission intensity at 342 nm (with the background intensity
from the buffer subtracted) was used for analysis and normalized by the
peak intensity without Ca2þ for each sample. The binding affinity and
stoichiometry of Ca2þ association with the LA TD was determined by
fitting the change in fluorescence intensity at 342 nm as a function of
Ca2þ concentration to a Hill model. For our system where the ligand is
the Ca2þ and the conformational change is assumed to be proportional
to the change in fluorescence intensity at the emission peak,
DI (342 nm), normalized by the peak emission at 342 nm without Ca2þ
in the solution, Io, the Hill equation is DI/Io ¼ [Ca2þ]n/(KDþ[Ca2þ]n)
gives a binding affinity of the protein to calcium KD, as well as the
stoichiometry of binding, n (i.e., the number of Ca2þ that associate with
one protein molecule).
MATERIALS AND METHODS
Protein production, farnesylation,
and characterization
Membrane bilayer preparation and surface
plasmon resonance experiments (SPR)
Constructs for the expression of LA TDs were created using polymerase
chain reaction from plasmids kindly provided by Misteli at NCI/NIH
(12). Plasmids were expressed in Escherichia coli, purified using glutathione-s-transferase fusion peptides and farnesylated in vitro using farnesyltransferase, resulting in a population of ~50% farnesylated protein (see
Methods in the Supporting Material). Purified proteins were verified for
size analysis using liquid chromatography electron spray ionization mass
spectroscopy, for purity using 14% SDS-PAGE, for aggregation by dynamic
light scattering (DLS) (nanosizer). Bradford assay (Coomaise Plus, Pierce)
was used to determine protein concentration (see Methods).
SPR was used to quantify the membrane association of the LA-TD to tethered bilayer lipid membranes (tBLMs), which provide a fluid membrane
fixed to a surface for SPR to monitor membrane association without the
necessity of using a protein label. The tBLM model membrane system
consists of a planar lipid bilayer suspended to the surface by a minimal
number of functionalized lipopolymer interspersed with b-mercaptoethanol to create a submembrane hydration layer (see Methods). The
~1.5 nm hydration layer allows the membrane to retain its fluidity and
reduce the effects of the substrate on the membrane. See Methods for
full details. The molecular structure, functionality, and in-plane dynamics
of such tBLMs have been established by the Lösche group in extensive
studies involving electrochemical impedance spectroscopy, neutron scattering, fluorescence microscopy, and fluorescence correlation spectroscopy
(22–24).
Solution conditions that minimize aggregation
of D50LA and preLA TDs
Recombinant LA-TD variants aggregate in water, therefore accurate quantification of protein-membrane interactions required stabilization of monomer protein. We determined the minimal salt (NaCl) concentrations in the
buffer necessary to suppress aggregation of D50LA-TD and preLA-TD by
DLS. Although deconvoluted DLS spectra were not able to determine
aggregation form, it clearly showed large-scale aggregation under lowsalt conditions (Fig. S1 in the Supporting Material). Characteristic DLS
peaks of d ~10 nm were regularly observed under high-salt conditions
([NaCl] > 50 mM), and we interpreted a 10 nm peak as a protein monomer.
At low salt ([NaCl] < 50 mM), proteins aggregated (d > 100 nm). This
analysis showed different minimal salt concentrations for the different
lamin A variant TDs studied here, including with farnesylation(fn).
D50LA-TD required 25 mM NaCl to maintain a monomeric solution of protein, whereas fn-D50LA-TD required 50 mM NaCl (Fig. S1 A). In contrast,
preLA-TDs required a minimum of 500 mM NaCl to maintain dispersed
solutions (Fig. S1 B). The addition of 2 mM CaCl2 did not alter the stability
of D50LA-TD and fn-D50LA-TD (stable in 50 mM NaCl and 2 mM CaCl2,
Fig. S1 C) or preLA-TD and fn-preLA-TD (stable in 500 mM NaCl and
2 mM CaCl2, Fig. S1 D). Aggregation could also be suppressed in
10 mM sodium phosphate buffer but could not be used in these experiments
because phosphate chelates calcium. Other salts, such as KCl or NaHCO3,
were not tried as membrane behavior under those conditions.
Tryptophan fluorescence
The tryptophan amino acid residue has inherent fluorescent properties that
have long been used as probes to reflect protein conformation (20). LA-TD
Replica exchange molecular dynamics (REMD)
simulations
Protein structure predictions were carried out by the REMD method (25)
implemented in the CHARMM c35b1 (26) software. Replica management
and conformation analysis are done using the MMTSB script package (27).
REMD is a simulation method with atomic detail to improve the dynamic
properties of the Monte Carlo method, aiming at a global-minimum free
energy state. It samples a wide conformation space without getting trapped
in local-minimum free energy states because of the thermal stimulation by
high-temperature replicas, and we can find stable structures at the replica
with the lowest temperature. The REMD inputs include the peptide
sequence and an initial guess of conformation (see Methods).
Modeling of farnesyl diphosphate
The structure and interaction scheme of the farnesyl diphosphate is not
defined in the standard CHARMM27 force field. We setup the initial
conformation of farnesyl diphosphate according to its atomic coordinates
as found in the protein data bank (with PDB ID 1KZO). The interactions
(covalent bond, angle, dihedral, electrostatic, and vdW interactions) among
the atoms in farnesyl diphosphate are defined in the CHARMM General
force field 2b6 (28). We updated the CHARMM27 force field to incorporate
the section of the definition of farnesyl diphosphate (see Methods for details
of the modeling and validation against the atomic structures identified from
experiment).
Biophysical Journal 104(10) 2246–2253
2248
Kalinowski et al.
RESULTS
the high salt required to maintain monomeric stability of
fn-preLA-TD (see Methods) interfered with membrane
stability, so that fn-preLA-TD binding to membranes could
not be quantified.
Membrane binding of fn-D50LA-TD depends on
calcium
To determine the membrane affinity of fn-D50LA-TD, we
added purified monomeric farnesylated protein (see
Methods and Supporting Material information for production, characterization, and monomeric stability) to tBLMs
and measured binding using SPR. The tBLMs are appropriate for precise SPR binding measurements because bilayers are virtually defect free, thus avoiding nonspecific
protein adsorption (29) and tightly coupled to gold-coated
solid substrates via hydrated oligo(ethylene oxide) spacers
(30). Bilayer membranes were composed of dioleoyl-phosphatidylcholine and the charged dioleoyl-phosphatidylserine (DOPC/DOPS ¼ 7:3) with 3 mol% cholesterol to
mimic lipid composition and charge of cellular membranes.
With or without farnesylation, D50LA-TD associated
minimally with the membrane in NaCl buffer (Fig. 1 A).
However, the presence of physiological levels of free
Ca2þ in the buffer facilitated protein-membrane interaction.
fn-D50LA-TD bound stably to tBLMs with 2 mM CaCl2 in
the buffer, but D50LA-TD binding was not detected under
otherwise identical conditions (Fig. 1, A and B). Increasing
concentrations of fn-D50LA-TD in the Ca2þ-containing
buffer increased membrane binding (Fig. 1 C) and showed
a binding affinity, KD ~2 mM (assuming that only the farnesylated proteins associated with the membrane, i.e., approximately half the concentration of the protein added) when fit
to a Langmuir model (Fig. 1 C). We note that the membrane
association is a combination of binding and aggregation,
which impacts the KD: instantaneous high levels of
fn-D50LA-TD do not show the same levels of binding as
increased amounts of fn-D50LA-TD (Fig. 1 B vs. Fig. 1
C). Furthermore, protein is not easily dissociated from the
interface. These and other aspects of interfacial aggregation
and binding are the topic of future work. Unfortunately,
0
0
1
0
1F
0.1F
3
Time, hr
2F
6
D
+Ca2+
200
100
0
0 .01F .05F
2
0.1F
0.5F
1F
Density (ng/cm2)
100
C
Density (ng/cm2)
To investigate the mechanism by which Ca2þ induces membrane association, we considered Ca2þ-induced conformational changes of the TD. The TD is intrinsically
disordered except for the s-type Ig-fold (31,32), therefore,
direct structural examination via traditional methods (e.g.,
crystallography) are not possible. Conformational changes
of D50LA-TD and preLA-TD were followed by monitoring
the fluorescence of their four Trp residues located within
and near the Ig-fold (W467, W498, W514, W520; see full
sequence in Table S1). The chemical environment of Trp,
including its solvent accessibility, affects the emission quantum yield, and exposure to the solvent has been shown to
quench Trp fluorescence (20,21). Previously, we investigated thermal denaturation of LA-TD and D50LA-TD tertiary structure by monitoring Trp fluorescence, confirming
that protein denaturation reduces Trp fluorescence (7).
The observed fluorescence emission band between 310
and 400 nm was broad, presumably because multiple Trp
residues in different microenvironments contribute to the
band shape. We observed that the emission decreased with
CaCl2 concentration (Fig. 2, A and B A).), and saturated for
[CaCl2] z 1 mM at ~30% of the original intensity. Fitting
to a two-parameter Hill model indicated a Ca2þ affinity of
261 5 49 mM for D50LA-TD and 286 5 27 mM for preLA
TD, both with 1:1 stoichiometries (510%) (Fig. 2 C). These
results suggest that Trp residues of the two TDs become more
exposed to buffer upon Ca2þ binding. High concentrations of
NaCl (>200 mM) also reduced Trp emission (Fig. S2), but
Ca2þ-induced effects occur at six orders of magnitude lower
salt concentration (100 mM), independent of NaCl.
B
−Ca2+
200
2F
6
Time, hr
Biophysical Journal 104(10) 2246–2253
10
Protein adsorbed
(saturated fraction)
Density (ng/cm2)
A
Calcium-dependent conformational changes
+Ca2+
200
100
0
0
0
1.5
3.2F
4
1
2
Time, hr
1
+Ca2+
0.5
0
−2
10
−1
10
0
10
1
10
[fn−Δ50LA−TD], μM
FIGURE 1 Binding of fn-D50LA-TD to a model
membrane. (A) 1 mM D50LA-TD (gray) did not
associate with the tBLM in 50 mM NaCl. Furthermore, 0.1 mM, 1 mM, or 2 mM fn-D50LA-TD in
50 mM NaCl (0.1 F, 1 F, and 2 F in blue) did not
associate with the membrane (B) For a similar
tBLM and buffer conditions plus 2 mM CaCl2,
1.5 mM or 4 mM D50LA-TD (gray) did not associate with the membrane. However, 3.2 mM
fn-D50LA-TD (3.2 F in red) did show association.
(C) Stepwise addition to the solution phase of
0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, and
2 mM fn-D50LA-TD (red) showed increased membrane association with 50 mM NaCl and 2 mM
CaCl2. (D) From this association we can produce
a binding curve to determine the KD of the protein
to the membrane. (tBLM composition: 30:70:þ3
DOPS/DOPC/Chol)
Farnesylated Lamin A Tail Domain
2249
I/I0 (a.u.)
A
B
Δ50LA−TD
1.0
increasing
2+
[Ca ]
0.5
315
C
355
λ (nm)
preLA−TD
1.0
increasing
2+
[Ca ]
0.5
395
315
355
λ (nm)
ΔI/I0 (λ = 342 nm)
1
2
R = 0.99
0.5
Δ50LA−TD
preLA−TD
0
−1
10
0
log [Ca2+], mM
10
Fluctuations of the Ig-fold of D50LA-TD
and preLA-TD in the presence of calcium
To obtain a deeper insight into the structural responses of the
LA TDs to Ca2þ, we performed molecular dynamics (MD)
simulations of the proteins in buffer. To deal with the high
degree of intrinsic disorder, we simulated multiple lowenergy, pseudoequilibrated structures with REMD (25,33).
Starting with amino acid chains with extreme initial structures (straight or fully coiled) except for the structured
Ig-fold region taken from the NMR structure in the protein
database (31), we allowed the protein to relax freely in an
implicit solvent environment. To enhance the sampling
speed as well as to ensure that structures reached relevant
local energy minima, we investigated numerous independent structures with implicit-solvent REMD to find as
many low-energy states as possible (see Methods). Results
were categorized according to their structural similarity,
and the four most significant conformations were chosen
for further studies of the structural impact of ions (7). These
four conformations were further refined in REMD simulations with explicit solvent under various ion conditions
(see Methods). To quantify the TD’s structural response to
ion exposure, we focused on four peripheral amino acids
(S429, S437, L478, and I497), which form orthogonal extremes of the two b-sheets that form the Ig-fold (Fig. 3 A).
From the positions of these four amino acids, we determined
an orientation angle between the two b-sheets, q (Fig. 3 A).
The Ig-fold is slightly more open (q is systematically larger)
in the presence of Ca2þ (Fig. 3 B).
We computationally included a farnesyl group to the
LA-TDs. For an initial conformation, we used the pseudoequilibrium structure of the unfarnesylated LA-TDs and
embedded the added C-terminal farnesyl group within the
hydrophobic core of the Ig-fold (see Methods for force
field). We then allowed the new structure to come to a
new equilibrium with explicit solvent. The Ig-fold of both
D50LA-TD and preLA-TD were more open if farnesylated,
395
FIGURE 2 Calcium caused a conformational
change to D50LA-TD and preLA-TD. The emission spectra of tryptophan residues for purified
(A) D50LA-TD in 50 mM NaCl and (B) preLATD in 500 mM NaCl excited at 295 nm were
measured for increasing Ca2þ concentration. For
Ca2þ concentration above 1 mM, there was no
change in intensity, indicating no more substantial
conformational change induced by Ca2þ. (C) From
plots of emission intensity at 342 nm versus Ca2þ
concentration, we determined Ca2þ dissociation
constant (KD ~250 300 mM) and binding stoichiometry (1:1). The DI/I0 (342 nm) is averaged
for 2–3 samples and in NaCl concentrations
50–700 mM NaCl for a given CaCl2 concentration.
The Ca2þ dissociation constant did not change with
the NaCl concentration of the solution.
and exposure to Ca2þ opened the fold further up by a larger
angle than with the unfarnesylated proteins (Fig. 3 C).
Because the average distance between I497 and L478
FIGURE 3 Altered Ig-fold structures of preLA-TD and D50LA-TD
exposed to calcium. (A) We determined the dihedral angle between the
two b-sheets of the Ig-fold, q, from the a-carbon of orthogonal amino acids
(S429, S437, I497, and L478) within the full TD. (B) We tracked the fluctuations of the dihedral angle q for the preLA-TA conformation in the
replica with lowest temperature after each exchange event in pseudoequilibrium. The angle was always larger in the presence of Ca2þ. (C) Statistical
comparison of the dihedral angle of the Ig-fold for numerous pseudoequilibrated protein structures for preLA-TD, fn-preLA-TD, D50LA-TD, and
fn-D50LA-TD showed increases in q with Ca2þ.
Biophysical Journal 104(10) 2246–2253
2250
is >1.7 nm, an increase in the orientation angle q indicates
that the space between the two b-sheets accommodates
more water (with a van der Waals radius of 0.14 nm) in
the presence of the ion than in its absence.
Kalinowski et al.
A
Fluctuations of the C-terminus of D50LA-TD
and preLA-TD with calcium
REMD simulations also provided insight into the mechanism by which calcium triggers membrane binding of farnesylated lamin A tail domains. We observed that Ca2þ bound
spontaneously to the C-terminal CaaX motif, and the interaction persisted through the entire REMD simulation (cyan
Ca2þ to red CSIM in Fig. 4 A, Fig. S3). Generally, Ca2þ
binding increased the fluctuation and mobility of the
C-terminal region for both D50LA-TD (beyond 6100 ; the
prime refers to the new amino acid number for the deletion)
and preLA-TD (beyond 660) by 30% and 39%, respectively
(Fig. 4 B).
C-terminal motility was reduced when the protein was
farnesylated (Fig. 4 B), and the Ca2þ-mediated motility
increase was enhanced for farnesylated proteins (Fig. 4 B)
by 89% for fn-preLA-TD and 120% for fn-D50LA-TD. In
the absence of Ca2þ, the farnesyl (Fig. 5, red) maintained
association with the hydrophobic core (Fig. 5 A, Ca2þ).
In the presence of Ca2þ, the farnesyl group escaped
the hydrophobic interior of the protein and was found at
the periphery of the pseudoequilibrated structures (Fig.
5 A, þCa2þ). This was quantified by the increase in solvent
available surface area of the farnesyl group in the presence
of Ca2þ (Fig. 5 B) and increased end-to-end length of the
TDs (Fig. 5 C). We suggest that the farnesyl group associates with the hydrophobic protein core, and the association
of Ca2þ to the CaaX causes increased probability of farnesyl
solvent exposure for membrane association.
DISCUSSION
Membrane binding of farnesylated lamin A tail
requires calcium
We find that fn-D50LA-TD binds to purified membrane
bilayers with a KD ~2 mM. This binding coefficient is markedly weaker than most biological binding coefficients; specific protein-protein binding typically occurs on the order
of 10s to 100s of nM. We suggest that this weak binding
affinity allows protein-membrane association while not
causing irreversible association. In the case of lamin A,
we suggest that the farnesyl group weakly associates the
protein to the membrane, and transmembrane lamin-binding
proteins then more permanently connect lamin A to the
inner nuclear membrane. This weak binding is similar to
qualitative observations that farnesyl groups are insufficient
to stably associate proteins with membranes (16–18). During normal processing, lamin TDs, and other CaaX-domain
Biophysical Journal 104(10) 2246–2253
B
FIGURE 4 Dynamics of the C-terminus of preLA-TD and D50LA-TD
exposed to calcium. (A) We examined all pseudoequilibrated conformations
of unfarnesylated preLA-TD produced in explicit-solvent REMD simulations and acquired snaphopys of the lowest energy conformations
(overlaid). In the presence of explicit water and sodium, the C-terminus
CSIM (red) was located inside the preLA tail structure. With the addition
of Ca2þ (cyan), the C-terminus pointed away from the protein. (B) The
C-terminal amino acids for each of the four TDs for 10 ns of MD simulation
showed altered fluctuations or root mean-square displacement from initial
coordinates in the presence of Ca2þ. For each TD and ionic condition we
repeated the simulation for the four lowest energy conformations and statistically compared the results. For the last 3 to ~6 residues, Ca2þ caused a
substantial increase in amino acid fluctuation. The Ca2þ-induced fluctuation changes were more substantial for farnesylated proteins, which showed
reduced C-terminal fluctuations in the absence of Ca2þ.
proteins, are farnesylated, the aaX residues are cleaved by
the ZMPSTE-1 protease, and the terminus is methylated
in the nucleus (34). The proteolysis and methylation steps
may play an additional role in stabilizing membrane association by increasing hydrophobicity. However, there have
been conflicting reports regarding the additional stability
conferred by methylation (35–37).
The association of the fn-D50LA-TD is dependent on
Ca2þ. There have been previous studies examining qualitative membrane association of fn-preLA (38), but we
note that the buffer used contained the divalent cation
magnesium. Similarly, recoverin has a Ca2þ-induced
Farnesylated Lamin A Tail Domain
2251
FIGURE 5 Simulation of farnesyl dynamics of
preLA and D50LA TD. We preequilibrated the farnesyl group within the hydrophobic interior of the
respective TDs and allowed equilibration in
explicit solvent with 50 mM NaCl or 50 mM
NaCl þ 2 mM CaCl2. (A) Comparison of the
four most significant pseudoequilibrated structures
of fn-preLA-TD. With no Ca2þ the farnesyl group
(red) was found within the molecule. In the presence of Ca2þ, the farnesyl group escaped from
the TD. (B) The solvent accessible surface area
of the farnesyl group, given by the number of
water molecules in contacting with the surface of
farnesyl group (threshold distance of 0.38 nm),
of fn-D50LA-TD and fn-preLA-TD increased in
Ca2þ. (C) The end-to-end length of the entire TD
of fn-D50LA-TD and fn-preLA-TD also increased,
suggesting that the Ca2þ makes the C-terminus
more extended.
conformational switch to expose the myristoyl group, which
is responsible for membrane association (39). Unlike recoverin, lamin A tail domains are mostly disordered, therefore
the conformational switch is much less obvious. We demonstrated through experiments and simulations that D50LATD and pre-LA TD undergo a conformational change
induced by Ca2þ that opens the Ig-fold and increases the
exposure and mobility of the farnesylated C-terminus.
The Ca2þ-induced conformational change may also regulate protein-membrane interactions, protein-DNA, or protein-protein interactions. The large size, intrinsic disorder,
and Ig-fold binding pocket of the tail domain may all allow
for promiscuous binding and may be at least partially
responsible for the ability of lamin A tail to bind different
partners (1). Here, we have shown Ca2þ-induced conformational changes, and protein binding to the lamin A of one of
the many lamin A-associated proteins may also induce
conformational changes in the tail domain. We find that,
similar to Ca2þ, Mg2þ can facilitate the membrane association of fn-D50LA-TD, and is the subject of future work.
Membrane association of farnesylated lamin A tail
domain includes binding and aggregation
Simulations of the D50LA-TD structure show the protein
diameter to be 5.2 5 0.2 nm, and a complete monolayer
of protein coverage is reached at ~1 mM of solution protein
concentration. However, above 1 mM we still observe additional, stable membrane association. This additional association is likely due to a combination of aggregation coupled
with binding. Because high salt levels are required to prevent aggregation of fn-preLA-TD, and proteins show no
membrane association as monomers, we suggest that the
protein-protein association of fn-preLA-TD overwhelms
the protein-membrane association with excess protein
(40). This highlights that fn-D50LA-TD has an apparent
greater affinity for the membrane and enhanced membrane
association of D50LA may contribute to the pathological
membrane association (12,13).
Lifetime of wild-type lamin A and pathological
D50LA
Most contemporary literature suggests that prelamin A farnesylation is responsible for the association of protein to the
inner nuclear membrane for further incorporation into the
lamina (10,11). It is unclear why lamins do not embed themselves into membrane structures outside the nucleus because
farnesyltransferases have been located in the cytoplasm near
the endoplasmic reticulum and inside the nucleus.
From the results presented here, we suggest changes in
the tail domain of preLA from the time it is produced
from LMNA mRNA until it is processed to form mature
lamin A. We speculate that after transcription, the C-terminal CaaX domain is hidden inside the TD of preLA. The
preLA (probably as a dimer) is transported into the nucleus
via the TD nuclear localization sequence (NLS). In the
nucleus, the higher levels of charge from DNA and counter
ions induce a conformational change that exposes the
CaaX for farnesylation and other modifications leading to
Biophysical Journal 104(10) 2246–2253
2252
insertion into the inner nuclear membrane. There are high
levels of divalent cations including calcium, magnesium,
and zinc inside the nucleus (41), but exact levels of free
divalent cations in the nucleus are difficult to determine
because free ions are buffered by surrounding chromatin
structure. It is also possible that small amounts of preLA
may be farnesylated by cytoplasmic farnesyltransferases.
We suggest that these farnesyl groups may incorporate
into the Ig-fold until they are exposed to high Ca2þ levels.
Ig-folds and similar b-sheet structures have been shown to
act as solubilization agents for lipidated proteins (42–44).
In the case of fn-preLA, the farnesylation could associate
with its own Ig-fold or that of a neighbor.
SUPPORTING MATERIAL
Five figures and supplemental methods are available at http://www.
biophysj.org/biophysj/supplemental/S0006-3495(13)00445-1.
Kalinowski et al.
11. Lutz, R. J., M. A. Trujillo, ., M. Sinensky. 1992. Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A
assembly into the nuclear lamina. Proc. Natl. Acad. Sci. USA. 89:3000–
3004.
12. Dahl, K. N., P. Scaffidi, ., T. Misteli. 2006. Distinct structural and
mechanical properties of the nuclear lamina in Hutchinson-Gilford
progeria syndrome. Proc. Natl. Acad. Sci. USA. 103:10271–10276.
13. Goldman, R. D., D. K. Shumaker, ., F. S. Collins. 2004. Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome. Proc. Natl. Acad. Sci.
USA. 101:8963–8968.
14. Reddy, S., and L. Comai. 2012. Lamin A, farnesylation and aging. Exp.
Cell Res. 318:1–7.
15. Kubben, N., M. Adriaens, ., T. Misteli. 2012. Mapping of lamin Aand progerin-interacting genome regions. Chromosoma. 121:447–464.
16. Silvius, J. R., and F. l’Heureux. 1994. Fluorimetric evaluation of the
affinities of isoprenylated peptides for lipid bilayers. Biochemistry.
33:3014–3022.
17. Nadolski, M. J., and M. E. Linder. 2007. Protein lipidation. FEBS J.
274:5202–5210.
18. Philips, M. R. 2012. Ras hitchhikes on PDE6d. Nat. Cell Biol.
14:128–129.
We gratefully acknowledge the assistance by Matthew Biegler (CMU MSE)
and Peter Yaron (CMU ChemE) and we are grateful to Katherine Wilson
and Susan Michaelis (Johns Hopkins Department of Cell Biology) for careful reading of the manuscript.
19. Taimen, P., K. Pfleghaar, ., R. D. Goldman. 2009. A progeria mutation reveals functions for lamin A in nuclear assembly, architecture,
and chromosome organization. Proc. Natl. Acad. Sci. USA.
106:20788–20793.
Z.Q. and M.J.B. received support from National Science Foundation (NSF)
(CMMI-0642545) and Office of Naval Research-Presidential Early Career
Award for Scientists and Engineers (ONR-PECASE) (N00014-10-1-0562).
K.N.D. and A.K. received support from the Progeria Research Foundation,
NSF (CBET- 0954421 CAREER) and National Institutes of Health (NIH)
National Institute on Aging (NIA) (NRSA F30-AG030905 to A.K.). M.L
received support from NIH (1R01-GM101647).
20. Vivian, J. T., and P. R. Callis. 2001. Mechanisms of tryptophan fluorescence shifts in proteins. Biophys. J. 80:2093–2109.
21. Chen, Y., and M. D. Barkley. 1998. Toward understanding tryptophan
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22. Shenoy, S., R. Moldovan, ., M. Losche. 2010. In-plane homogeneity
and lipid dynamics in tethered bilayer lipid membranes (tBLMs). Soft
Matter. 2010:1263–1274.
23. Vockenroth, I. K., C. Ohm, ., I. Köper. 2008. Stable insulating tethered bilayer lipid membranes. Biointerphases. 3:FA68–FA73.
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