proteins
STRUCTURE O FUNCTION O BIOINFORMATICS
Two-state conformational equilibrium in the
Par-4 leucine zipper domain
Martin Schwalbe,
1
Kaushik Dutta,
2
David S. Libich,
1
Hariprasad Venugopal,
1
Jolyon K. Claridge,
1
David A. Gell,
3
Joel P. Mackay,
3
Patrick J. B. Edwards,
1
and Steven M. Pascal
1
*
1
Centre for Structural Biology, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
2
New York Structural Biology Centre, 89 Convent Avenue, New York, New York 10027
3
School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia
INTRODUCTION
Prostate apoptosis response factor-4 (Par-4) is a pro-apoptotic and tumor-sup-
pressive protein that is ubiquitously expressed and evolutionary conserved.
1–3
Initially identified as a pro-apoptotic factor upregulated in prostate cancer cells
undergoing apoptosis,
1,4
Par-4 was also found to function in various other can-
cers.
5
In contrast, upregulation of Par-4 has been detected in neurons linked with
various neurodegenerative diseases.
5
Amino acid sequence analysis predicts the N-terminal regions of Par-4 to be
mainly disordered,
6
whereas the C-terminus (residues 292–332 of racine Par-4) is
predicted to form a leucine zipper (LZ), based on a highly conserved heptad
repeat.
5
This LZ domain was shown to be essential for the sensitization of some
cancer cells to apoptotic stimuli.
4
Further, the LZ is required for self-associa-
tion
6,7
and binding to potential effector molecules
3,8
(for a more extensive list of
effector molecules see also Ref. 6). Some of these interactions have been shown to
be important for the pro-apoptotic and tumor-suppressive activity of Par-4.
3
The
LZ is part of a larger coiled coil region within the C-terminus of Par-4 (residues
248–332).
6
This coiled coil region shares some homology with the death domains
of other apoptotic proteins.
7,9
Coiled coils are common oligomerization motifs and are predicted in
10%
of all eukaryotic proteins.
10
These amphipathic a-helical motifs are able to form
left-handed supercoiled structures by wrapping two or more helices around each
other.
11
The amino acid sequence of coiled coils is characterized by a seven resi-
due repeat designated (abcdefg)
n
.
12
The a and d positions of each heptad are
generally hydrophobic and form the centre of the oligomeric interface. Amino
acids at other positions are usually polar or charged.
Coiled coils are associated with various functions and can occur either as
homo- or hetero-complexes. Short coiled coils of six or seven heptad repeats
often function as protein–protein interaction domains in signal transduction
pathways.
13–19
Long coiled coils of several hundred residues can form fibers,
Abbreviations: CCD, coiled coil dimer; CSI, chemical shift index; DLS, dynamic light scattering; DTT, dithiothre-
itol; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; GST, glutathione-S-transferase; LZ, leucine zipper;
MALLS, multi angle laser light scattering; NMR, nuclear magnetic resonance; Par-4, prostate apoptosis response
factor 4; POM, partially ordered monomer; RP-HPLC, reversed phase-high performance liquid chromatography;
SEC, size exclusion chromatography.
Additional Supporting Information may be found in the online version of this article.
Grant sponsor: Royal Society of New Zealand (Marsden Fund Award); Grant number: MAU0507.
*Correspondence to: Steven Pascal, Institute of Fundamental Sciences, Massey University, Turitea Site, Private Bag
11222, Palmerston North 4442, New Zealand. E-mail: s.pascal@massey.ac.nz.
Received 13 December 2009; Revised 1 April 2010; Accepted 5 April 2010
Published online 15 April 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.22752
ABSTRACT
Prostate apoptosis response factor-4
(Par-4) is a pro-apoptotic and tu-
mor-suppressive protein. A highly
conserved heptad repeat sequence at
the Par-4 C-terminus suggests the
presence of a leucine zipper (LZ).
This C-terminal region is essential
for Par-4 self-association and inter-
action with various effector proteins.
We have used nuclear magnetic reso-
nance (NMR) spectroscopy to fully
assign the chemical shift resonances
of a peptide comprising the LZ do-
main of Par-4 at neutral pH. Further,
we have investigated the properties
of the Par-4 LZ domain and two
point mutants under a variety of
conditions using NMR, circular
dichroism (CD), light scattering, and
bioinformatics. Results indicate an
environment-dependent conforma-
tional equilibrium between a par-
tially ordered monomer (POM) and
a predominantly coiled coil dimer
(CCD). The combination of techni-
ques used allows the time scales of
the equilibrium to be probed and
also helps to identify features of the
amino acid sequence that may influ-
ence the equilibrium.
Proteins 2010; 78:2433–2449.
VC 2010 Wiley-Liss, Inc.
Key words: prostate apoptosis response
factor 4; leucine zipper; circular dichro-
ism; solution NMR spectroscopy.
VC 2010 WILEY-LISS, INC. PROTEINS
2433

meshworks, or scaffolds because of their rod-like tertiary
structures
11
and have various functions, most notably as
components of the cytoskeleton,
20
the extra cellular ma-
trix,
21
and cellular motor proteins.
22
The LZ is a special
case of the coiled coil having predominantly leucine at
position d in each heptad repeat. LZs typically function
as dimerization or oligomerization domains and are of-
ten found in transcription factors.
15,16
Coiled coil formation by a peptide comprising the Par-
4 LZ domain (residues 286–332) was shown to be pH,
temperature, and concentration dependent.
23
The pH
dependency was attributed to the electrostatic repulsion
between two acidic residues at position e and g.TwoPar-
4 LZ point mutants, where these key acidic residues had
been mutated to lysines, displayed a pH-independent
coiled coil formation.
24
Here, we show that the Par-4 LZ
domain is in conformational exchange between a par-
tially ordered monomer (POM) and a more ordered, pre-
dominantly coiled coil dimer (CCD). The CCD displays
typical features of a coiled coil by circular dichroism
(CD) spectropolarimetry, however, nuclear magnetic res-
onance (NMR) spectroscopy suggests a degree of instabil-
ity that is related to peculiarities within the Par-4 LZ
sequence. The observed conformational exchange
between the POM and CCD conformations is slow on an
NMR time scale. Considering that the Par-4 LZ domain
is essential for binding to some of Par-4
0
s effector mole-
cules,
3,8
coiled coil destabilization may disfavor homo-
oligomerization of full-length Par-4 under certain condi-
tions, facilitating association with binding partners.
MATERIALS ANDMETHODS
Polymerase chain reaction (PCR) primers were pur-
chased from Operon Technologies (Ebersberg, Germany).
Restriction enzymes were purchased from Roche Diagnos-
tic GmbH (Penzberg, Germany). D
2
O and
15
NH
4
Cl were
obtained from Cambridge Isotope Laboratories (Andover,
MA). All other chemicals were of reagent grade or higher.
Expression of Par-4
The wild-type leucine zipper domain (rrPar-4LZ) of
Par-4 comprising residues 286–332 (racine sequence
numbering) was prepared by PCR amplification using
the primers 5
0
-GCACTAGGATCCCAAGATAAAGAA
GAAATG-3
0
(forward) and 5
0
-GCACTAGGATCCCTAAA
GAGTTTTATTTTCCTG-3
0
(reverse). As a template for
the PCR, a previously described wild-type Par-4 leucine
zipper construct in the HMBP-3C vector was used.
23
The PCR product was then cloned into the BamHI/SalI
sites of pGex-6P-3 (GE Healthcare, Uppsala, Sweden).
Preparation of the LZ domain point mutants rrPar-
4LZD305K and rrPar-4LZE310K in HMBP-3C has been
described previously.
24
All three Par-4 expression vectors were transformed into
E. coli BL21(DE3) CodonPlus cells (Stratagene, La Jolla,
CA). Typically, isotopic labeling was achieved by growing
the cells in Luria-Bertani medium at 378C until OD
600
5
0.6. Cells were harvested by centrifugation and resus-
pended in half the original volume of M9 minimal media
with
15
NH
4
Cl as the sole nitrogen source. The samples
used for chemical shift assignment were expressed simi-
larly, but also contained uniformly
13
C-labeled glucose as
the sole carbon source. After growing the cells for 1 h at
308C, expression was induced by the addition of isopropyl-
b-D-thiogalactopyranoside to a concentration of 0.4 mM,
and cells were grown for a further 6 h at 258C.
The LZ domain point mutants rrPar-4LZD305K and
rrPar-4LZE310K in HMBP-3C were purified as follows.
Cells were harvested by centrifugation, resuspended in lysis
buffer (50 mM Tris, pH 8.0, 100 mM NaCl, and 25 mM
imidazole), and lysed by three passes through a French
Press. The resulting lysate was clarified by filtration
through a 0.8 lm syringe filter and the Par-4 fusion pro-
teins were purified by Ni-NTA chromatography (GE
Healthcare, Uppsala, Sweden). Par-4 fusion proteins were
eluted with lysis buffer containing 250 mM imidazole and
subsequently dialyzed against lysis buffer. Par-4 fusion pro-
teins were cleaved at 48C to completeness with recombi-
nant 3C protease, purified as described previously,
25
and
passed again over the Ni-NTA column. Par-4 containing
fractions were pooled and concentrated using a Vivaspin
20 device (Vivascience AG, Hannover, Germany).
Because of solubility limitations, the rrPar-4LZ was
purified under denaturing conditions. Cells were resus-
pended in lysis buffer [20 mM Tris, pH 8.0, 8 M urea,
250 mM NaCl, and 5 mM dithiothreitol (DTT)] and
lysed by two passes through a French Press. The resulting
lysate was clarified by filtration through a 0.8 lm syringe
filter. The cleared lysate was stepwise dialyzed to 20 mM
Tris, pH 8.0, 150 mM NaCl, and 1 mM DTT, each step
reducing the concentration of urea and NaCl. The Par-4
fusion protein was purified with Glutathione Sepharose
(GE Healthcare, Uppsala, Sweden) according to manufac-
turer instructions. GST (glutathione-S-transferase)-Par-4
fusion protein was eluted with 50 mM Tris, pH 8.0, 10
mM reduced glutathione, and subsequently dialyzed
against 20 mM Tris, pH 8.0, 150 mM NaCl, and 1 mM
DTT. The fusion protein was cleaved to completeness
with 3C protease at 48C and passed again over the Gluta-
thione Sepharose column to remove the cleaved GST tag.
Par-4 containing fractions were pooled and concentrated
using a Vivaspin 20 device.
All Par-4 LZ peptides were further purified by reversed
phase-high performance liquid chromatography (RP-
HPLC) using a Delta-Pak C18 300 A
˚
column, 300 3 3.9
mm (Waters Corporation, Milford, MA), with a linear
water-acetonitrile gradient of 30–50% in the presence of
0.1% trifluoroacetic acid. Par-4 containing fractions were
pooled and lyophilized. The lyophilizate was resolubilized
M. Schwalbe et al.
2434
PROTEINS