Structure of the catalytic domain of the hepatitis C virus NS2-3 protease
(Supplementary Online Data)
Ivo C. Lorenz*, Joseph Marcotrigiano*,+ , Thomas G. Dentzer, and Charles M. Rice+
Laboratory of Virology and Infectious Disease
Center for the Study of Hepatitis C
The Rockefeller University
1230 York Avenue
New York, NY 10021, USA
*These authors contributed equally to this work.
+
To whom correspondence should be addressed
(ricec@rockefeller.edu, marcotj@rockefeller.edu)
Methods
Protein Expression and Purification
NS2 is a hydrophobic protein with several putative transmembrane segments1,2 (Fig. 1a).
Removal of the N-terminal region containing these segments greatly increases the expression and
yield of recombinant NS2 without affecting NS2-3 proteolysis3,4. A fragment encompassing amino
acids 94 to 217 of the NS2 coding sequence from HCV genotype 1a strain H77 (residues 903 to 1026
numbered according to the HCV polyprotein sequence) was cloned into a pET28a vector (Novagen).
Escherichia coli BL21 (DE3) transformed with the pET28a NS2 94-217 (NS2pro) plasmid were grown
at 30ºC until an OD (600 nm) of 1.0-1.1 was reached. The bacteria were then induced with 0.5 mM
isopropyl--D-thiogalactopyranoside and incubated at 18°C for 20 h. Cells were pelleted and
resuspended in lysis buffer, containing 50 mM Tris-HCl pH 7.5, 150 mM KCl, 10% glycerol, and 3%
Triton X-100. Following lysis by three passes through an Avestin Air Emulsifier, cell extracts were
clarified by centrifugation, and imidazole was added to a final concentration of 30 mM. The extracts
were then loaded on a HiTrap nickel immobilized metal affinity chromatography column
(Amersham/Pharmacia) equilibrated with buffer 1 (25 mM Tris-HCl pH 7.5, 150 mM KCl, 10%
glycerol, 3% Triton X-100, and 30 mM imidazole). Following extensive washing with buffer 1, the
detergent was exchanged by a linear gradient from buffer 1 to buffer 2 (50 mM Tris-HCl pH 7.0, 150
mM KCl, 10% glycerol, 1% n-octyl--glucopyranoside [nOG], and 30 mM imidazole). NS2pro was
eluted with buffer 2 supplemented with 300 mM imidazole. The N-terminal hexahistidine tag was
removed by overnight digestion with thrombin at 4ºC. Subsequently, the protein was loaded onto a
HiTrap SP cation exchange column (Amersham/Pharmacia) equilibrated with buffer 3 (50 mM MES
pH 6, 10% Glycerol, 1% nOG). After washing, the protein was eluted with a linear gradient from 0 to 1
M KCl. Fractions containing NS2pro were pooled and loaded onto a Sephacryl S-200 gel filtration
column (Amersham/Pharmacia) equilibrated with buffer 4 (50 mM MES pH 6, 150 mM KCl, 10%
glycerol, 5.4 mM n-decyl--maltopyranoside [DM]). After gel filtration, the protein was concentrated on
a Resource S cation exchange column (Amersham/Pharmacia) equilibrated with buffer 5 (50 mM
MES pH 6, 10% glycerol, 5.4 mM DM). Following washing, the protein was eluted with buffer 5
supplemented with 350 mM KCl. Final protein yields were typically between 1 and 2 mg per liter of
bacterial expression culture. The purity of the protein assessed by SDS-PAGE was greater than 95%.
Selenomethionine-labelled NS2pro was expressed in Escherichia coli BL21 (DE3) grown in minimal
medium supplemented with L-amino acids except methionine, and 50 µg/ml selenomethionine5, and
purified as described above.
2
Crystal Growth and Freezing
Crystals of NS2pro were grown by hanging drop vapour diffusion at 4C. The 500 microlitres of
well solution contained 100 mM Tris pH 8.5, 0.8 M ammonium acetate, 0.25 M lithium chloride and
12% (w/v) polyethylene glycol 3350. The drop consisted of 2 microliters of NS2pro at 6 to 9 mg/ml in 50
mM MES pH 6, 350 mM KCl, 10% glycerol, 5.4 mM DM, and 2 microliters of well solution. Cubic or
rhombic crystals of 0.1 to 0.2 mm in size grew from these conditions in approximately 6 days. For
freezing, crystals were transferred to well solution supplemented with 5.4 mM DM, with glycerol being
added stepwise to a final concentration of 25% over 45 minutes. Subsequently, crystals were
harvested and flash frozen in liquid propane.
Data Collection, Processing and Model Building
Data sets were collected at beamlines X9A and X29, National Synchrotron Light Source,
Brookhaven National Labs, Upton, NY. MAD data were collected at two wavelengths corresponding to
the peak (
2, 0.97939Å) of the selenium K absorption edge
(Table 1). A native data set was collected at a wavelength of 1.10000 Å. The datasets were
processed and scaled using DENZO/SCALEPACK6. Using the anomalous signal from the peak
selenomethionine data set, 19 of the 24 selenium sites were found using SnB7. An interpretable
electron density map was obtained using MLPHARE followed by density modification and phase
combination by SOLOMON and DM8. Several rounds of iterative model building and refinement were
performed using the programs O9 and CNS10. The native structure was determined by molecular
replacement with the program MOLREP8 using a search model containing the partially refined
structure from the selenium data sets.
The native and selenomethionine derivatized protein yielded two crystal forms in the identical
crystallization condition. Both forms had the same space group (P21),  angle, and two unit cell edges,
whereas the third axis of the native protein was twice as long as the derivatized form. The smaller unit
cell contained six NS2pro molecules per asymmetric unit, which were organized into three tightly
packed dimers. The larger unit cell was composed of twelve NS2pro molecules, arranged as two
hexamers containing three dimeric NS2pro molecules each (Supplementary Fig. 1). Despite the
apparent similarity of the two crystal forms, the orientation between the two trimers was different,
resulting in a larger unit cell for the native form of the protein. All molecules in the asymmetric unit
were similar with an r.m.s.d. ranging from 0.25 to 0.30 Å for corresponding alpha carbon positions
from different chains. The final refined model contains 176 solvent molecules, twelve detergent
molecules, twelve molecules of NS2pro (residues 94-217), and three additional N-terminal residues
(Ser-His-Met) remaining after thrombin digestion. A summary of the final refinement statistics is
provided in Supplementary Table 1. No unfavourable (,) combinations were found with
3
PROCHECK11, and main-chain and side-chain structural parameters were consistently better than or
within the average for structures refined to 2.3 Å.
Secondary structures were assigned using the program DSSP12. Graphics presented in this
manuscript were generated using the program PyMOL13. Atomic coordinates for the structures shown
in Figures 2c-f were obtained from the Protein Data Bank under accession numbers 1PPN (papain),
1L1N (poliovirus 3Cpro), 1KXF (Sindbis virus capsid), and 2SEC (Subtilisin/Eglin-C). GRASP14 and
LOOSENGRASP (D. Jeruzalmi, personal communication) were used for calculating surface
potentials. Sequence alignments were performed using ClustalX15, and plotting of conservation to
molecular surfaces was performed using the program msf_similarity_to_pdb (D. Jeruzalmi, personal
communication).
Crosslinking
Purified NS2pro at various concentrations (ranging from 2 to 100 µM) was mixed with 100 µM
disuccinimidyl suberate (DSS). After 2 hours incubation at room temperature, the proteins were
analyzed by SDS-PAGE, followed by staining of the gel with Coomassie Blue.
Transfection of Cells, Metabolic Labeling, and Cell Lysis
For the experiments with full-length HCV polyproteins, Huh-7.5 cells were infected with vTF7-3
helper virus expressing T7 polymerase as described previously16. After 1 hour incubation at 37ºC, the
cells were lipid-transfected with pBRTM plasmids coding for a full-length HCV polyprotein (coreNS5B) that contained NS2 with either wild-type His 143 and Cys 184, a single H143A or a single
C184A mutation, or a double H143A/C184A mutation. 16 hours post-transfection, the cells were
starved with cysteine/methionine-free medium for 30 minutes, followed by metabolic labeling using
35
S-cysteine/methionine (100 µCi/ml) for 1 hour. Cells were lysed in SDS lysis buffer supplemented
with protease inhibitors and processed as described previously16.
FLAG- and HA-tagged NS2-3 variants containing either wild-type, single-, or double-mutant
NS2 active sites (see above) were cloned into a pcDNA3.1(+) vector. U2OS osteosarcoma cells were
lipid-transfected with pcDNA3.1(+) plasmids coding for FLAG- or HA-tagged NS2-3 containing a wildtype or mutant NS2 active site. 24 hours post-transfection, the cells were starved, metabolically
labeled and lysed as described above. For the co-immunoprecipitation experiment (Fig. 3e), a lysis
buffer containing 1% CHAPS instead of SDS was used.
Immunoprecipitation
Cell lysates were incubated with anti-NS2 (WU10716), anti-NS2-NS3 (WU4316), anti-FLAG
(M2, Sigma) or anti-HA (HA.11, Covance) antibodies with end-over rotation at 4ºC overnight.
4
GammaBind plus Sepharose beads (Amersham) were added, followed by incubation with end-over
rotation at 4ºC for 2 hours. Immune complexes were washed three times with lysis buffer containing
detergent. After a final wash with lysis buffer that contained no detergent, SDS-PAGE sample buffer
was added to the immune complexes. The samples were heated at 95ºC for 5 min, centrifuged for 2
min and subjected to SDS-PAGE, followed by autoradiography.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Santolini, E., Pacini, L., Fipaldini, C., Migliaccio, G. & Monica, N. The NS2 protein of hepatitis
C virus is a transmembrane polypeptide. J Virol 69, 7461-71. (1995).
Yamaga, A. K. & Ou, J. H. Membrane topology of the hepatitis C virus NS2 protein. J Biol
Chem 277, 33228-34 (2002).
Pallaoro, M. et al. Characterization of the hepatitis C virus NS2/3 processing reaction by using
a purified precursor protein. J Virol 75, 9939-46. (2001).
Thibeault, D., Maurice, R., Pilote, L., Lamarre, D. & Pause, A. In vitro characterization of a
purified NS2/3 protease variant of hepatitis C virus. J Biol Chem 276, 46678-84. (2001).
Doublie, S. Preparation of selenomethionyl proteins for phase determination. Methods
Enzymol 276, 523-30 (1997).
Otwinowski, Z. & Minor, W. Processing of X-ray Diffraction Data Collected in Oscillation Mode.
Methods Enzymol 276, 307-26 (1997).
Weeks, C. M. & Miller, R. The design and implementation of SnB v2.0. J Appl Cryst 32, 120-4
(1999).
Collaborative Computational Project. The CCP4 suite: programs for protein crystallography.
Acta Crystallogr D 50 (Pt 5), 760-3 (1994).
Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein
models in electron density maps and the location of errors in these models. Acta Crystallogr A
47 (Pt 2), 110-9 (1991).
Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular
structure determination. Acta Crystallogr D 54 (Pt 5), 905-21 (1998).
Laskowski, R. A., Moss, D. S. & Thornton, J. M. Main-chain bond lengths and bond angles in
protein structures. J Mol Biol 231, 1049-67 (1993).
Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of
hydrogen-bonded and geometrical features. Biopolymers 22, 2577-637 (1983).
DeLano, W. L. The PyMOL Molecular Graphics System (www.pymol.org). (2002).
Nicholls, A., Sharp, K. & Honig, B. in Proteins: Structure, Function and Genetics 281-96
(1991).
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. The
CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by
quality analysis tools. Nucleic Acids Res 25, 4876-82 (1997).
Grakoui, A., McCourt, D. W., Wychowski, C., Feinstone, S. M. & Rice, C. M. A second
hepatitis C virus-encoded proteinase. Proc Natl Acad Sci U S A 90, 10583-7. (1993).
5