1 mM EDTA, and 5 mM DTT was mixed with an equal volume of reservoir
solution containing 28% (wt/vol) polyethylene glycol (PEG) 1500 and 100 mM
Tris·Cl (pH 8.0). A total of 7.5 mg/mL ERK2 D319N in 25 mM Tris·Cl (pH 7.5),
100 mM NaCl, 1 mM EDTA, and 5 mM DTT was mixed with an equal volume
of reservoir solution containing 1.5 M ammonium sulfate, 100 mM Hepes
(pH 7.75), and 2% (vol/vol) PEG 500 monomethyl ether. D319N crystals were
initially small needle clusters, but after 3 rounds of serial seeding, large plate
crystals were obtained. Crystals were incubated at 20 °C and harvested at
1 wk for E320K and at 3 d for D319N. Diffraction data were collected at
beamline 19-ID at the Advanced Photon Source (Argonne National Labora-
tory, Argonne, IL). Data were indexed, integrated, and scaled in HKL3000
(55). The structure of ERK2 E320K was determined using molecular re-
placement in PHASER in Phenix (56–58). The N- and C-terminal domains
(residues 6 to 169 and 170 to 358) of the active ERK2 structure (PDB ID code
2ERK) were used as search models, and a similar procedure using ERK2 wild
type (PDB ID code 4GBS) was employed for ERK2 D319N. The structures
contain 1 molecule per asymmetric unit. Iterative rounds of refinement (TLS,
rigid body, individual atomic displacement parameters, and atomic positions
[only N-terminal domain atomic positions for E320K]) were carried out in
phenix.refine, and model building was carried out in Coot (58–60). Sec-
ondary structure restraints were used early in model building for E320K.
Simulated annealing was used early in model building for D319N. Model
validation was conducted in phenix.refine, which uses analyses derived, in
part, from the MolProbity web server (58, 60, 61).
solution and 0.5% Triton-X were added, followed by sonication. Clarified
lysates were incubated with nickel-nitrilotriacetic acid beads (0.5 mL of
beads per liter of culture) for 1 h at 4 °C while rocking. All subsequent
washes were carried out in a conical tube, and beads were sedimented by
centrifugation. Beads were washed 5 times each with 10 bed volumes of
50 mM Na-phosphate, 300 mM NaCl, and 40 mM imidazole (pH 8.0). Proteins
were eluted by incubation with 3 bed volumes of 50 mM Na-phosphate (pH
8.0), 300 mM NaCl, and 300 mM imidazole (pH-adjusted) for 30 min at 4 °C
with occasional stirring by pipette. Before MonoQ purification, the sample was
filtered through a 0.2-μm filter and then dialyzed into 10 mM Tris·HCl (pH 8.2)
and 0.5 mM TCEP (buffer A). Filtering is essential to prevent aggregation during
dialysis into the low-salt buffer. Samples were passed over a MonoQ GL 5/5
column (GE Life Sciences) at 0.3 mL·min−1. Protein was eluted by 3 column
volumes of buffer A, followed by a 0 to 25% gradient of buffer B (buffer
A + 1 M NaCl) over 40 volumes. Western blotting using ppERK2 antibody was
used to determine which fractions to exclude due to autophosphorylation.
Protein is highly pure after MonoQ, but Superdex200 gel filtration was used to
prepare protein for biophysical measurements and crystallography. Gel fil-
tration buffer was 25 mM Tris·HCl (pH 7.6), 100 mM NaCl, 1 mM TCEP, and
1 mM EDTA.
Data and Materials Availability. The following crystallographic datasets were
used: ERK2 D319N (PDB ID code 6OT6) and ERK2 E320K (PDB ID code 6OTS).
All other materials are available upon request.
Dynamic Light Scattering. ERK2 dynamic light scattering measurements were
made using a DynaPro NanoStar (Wyatt Technologies). Samples purified by
Superdex200 gel filtration at 24 μM (1 mg/mL) in 25 mM Tris·HCl (pH 7.6),
100 mM NaCl, 1 mM TCEP, and 1 mM EDTA were used. Analysis was carried
out using the DYNAMICS software package.
ACKNOWLEDGMENTS. We thank Dominika Borek, Diana Tomchick, and Zhe
Chen (Department of Biophysics, UT Southwestern) for advice and assistance
with crystallization, data collection, and data processing; Chad Brautigam
and Shih-Chia Tso (Department of Biophysics, UT Southwestern) for assis-
tance with dynamic light scattering and microscale thermophoresis; Radha
Akella (Department of Biophysics, UT Southwestern) for assistance with
thermal stability assays; Dean Smith and Ben Weaver (Department of
Pharmacology, UT Southwestern) for thoughtful comments on the manu-
script; Michael Reese (Department of Pharmacology, UT Southwestern) for
suggestions about plotting B-factor data; Aroon Karra, Wen-Huang Ko,
Jihan Osborne, and other current and former members of the M.H.C. labo-
ratory for their advice and assistance with specific aspects of this project; and
Dionne Ware for administrative assistance. Results shown in this report are
derived from work performed at the Argonne National Laboratory, Struc-
tural Biology Center at the Advanced Photon Source. The Argonne National
Laboratory is operated by the University of Chicago Argonne, LLC, for the US
Department of Energy, Office of Biological and Environmental Research,
under contract DE-AC02-06CH11357. This project was supported by Welch
Foundation Grants I1243 (to M.H.C.) and I1128 (to E.J.G.) and by NIH Grants
R37DK34128 (to M.H.C.) and R01GM086537 (to S.Y.S.). C.A.T. was supported
in the late stages of this work by Cancer Prevention and Research Institute of
Texas Training Grant RP160157. Additional support was provided by Na-
tional Cancer Institute Grant P30CA142543 to the Harold C. Simmons Com-
prehensive Cancer Center for core facilities.
Thermal Stability. Temperature-dependent changes in SYPRO orange fluo-
rescence were monitored using a C1000 Thermal Cycler combined with a
CFX96 Real-Time Monitoring System (Bio-Rad). ERK2 protein was at 5 μM in
25 mM Tris·HCl (pH 7.6), 100 mM NaCl, 1 mM TCEP, and 1 mM EDTA. Tm was
defined as the temperature of maximum change in SYPRO orange fluores-
cence and determined in Microsoft Excel.
Protein Expression and Purification. His6-tagged rat ERK2 wild type and mu-
tants in a NpT7 vector were expressed in Escherichia coli DE3. Fifty-milliliter
starter cultures were grown overnight at 30 °C in the presence of 100 μg/mL
ampicillin in Luria broth. Twenty milliliters was then used to inoculate 1-L
cultures, which were grown at 30 °C. Protein expression was induced with
400 μM isopropyl-β-D-1-thiogalactopyranoside at an optical density at
600 nm = 0.7, and cultures were allowed to grow overnight at 30 °C (21 h
postinduction worked best). Cultures were centrifuged, and pellets were
frozen in liquid nitrogen. Pellets were thawed and resuspended in 50 mM
Na-phosphate (pH 8.0) and 300 mM NaCl. Ten percent saturated lysozyme
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