Two-state selection of conformation-
Junjun Gaoa, Sachdev S. Sidhu1,b, and James A. Wellsa,2
aDepartments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California, 1700 4th Street, San Francisco, CA 94143;
andbDepartment of Protein Engineering, Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080
Communicated by Robert M. Stroud, University of California, San Francisco, CA, December 22, 2008 (received for review October 19, 2008)
We present a general strategy for identification of conformation-
specific antibodies using phage display. Different covalent probes
were used to trap caspase-1 into 2 alternative conformations,
forms of the protease were used as antigens in alternating rounds
of selection and antiselection for antibody antigen-binding frag-
ments (Fabs) displayed on phage. After affinity maturation, 2 Fabs
were isolated with KD values ranging from 2 to 5 nM, and each
their noncognate conformer. Kinetic analysis of the Fabs indicated
that binding was conformation dependent, and that the wild-type
caspase-1 sits much closer to the off-form than the on-form.
Bivalent IgG forms of the Fabs were used to localize the different
states in cells and revealed the activated caspase-1 is concentrated
in a central structure in the cytosol, similar to what has been
described as the pyroptosome. These studies demonstrate a gen-
eral strategy for producing conformation-selective antibodies and
show their utility for probing the distribution of caspase-1 confor-
mational states in vitro and in cells.
allostery ? caspase-1 ? phage display ? protein conformational change
selection upon binding of different small molecules, biopoly-
mers, or metal ions or through posttranslational modifications.
Structural methods give us high-resolution insight into the
nature of these conformational transitions in vitro but have
limited use for determining the equilibrium distribution of these
states in solution or in cells. To expand the tools useful for
trapping and analyzing conformational states in enzymes, both
in solution and in cells, we developed a general strategy for
As a test case, we focused on caspase-1, an aspartate-specific
thiol protease that is critical for processing of proinflammatory
cytokines during the innate immune response (for review, see
exists primarily as a monomer in solution (4, 5). Upon innate
immune stimuli, the proenzyme is believed to dimerize by
binding to scaffolding proteins known collectively as the inflam-
masome. This triggers proteolytic autoactivation or transactiva-
tion, in which the propeptide and an intersubunit linker are
cleaved (6, 7). Crystal structures of the mature protease with
various small molecules bound show that it can exist in at least
2 conformations (8–10). When an active site inhibitor is bound,
the enzyme appears to be in a catalytically competent form
(called the on-form) (9). However, binding of covalent disulfide
ligand to a central cavity ?15 Å from the active site stabilizes the
protease in an inactive state (called the off-form) (8). This
allosterically inhibited state is virtually identical to the apo-form
of the enzyme as seen in the crystal structures. The dimeric
enzyme shows positive cooperativity [nhill? 1.5 (8)], and mu-
tational studies reveal that only a small set of residues (a
‘‘hot-wire’’) mediates the on-to-off transition between the allo-
rotein allostery is a central means to regulate protein func-
tion in cells. Allostery is mediated through conformational
steric and active sites (11). These data support a dynamic
activation model for caspase-1 (Fig. 1A).
We wished to generate monoclonal antibodies to each of the
on- and off-states to better understand the equilibrium distri-
bution of these states in solution and in cells and to provide
probes to localize these forms in cells. We trapped homogeneous
forms of the on- and off-states of caspase-1 using the active site
inhibitor (Ac-YVAD-cmk) to lock the on-state and compound
34 (1-methyl-3-trifluoromethyl-1H-thieno[2,3-c]pyrazole) to
lock the off-state. These conformation-locked forms of
caspase-1 were then used as antigens in sorting codon-restricted
phage display libraries (12) to generate high-affinity antibody
Author contributions: J.G., S.S.S., and J.A.W. designed research; J.G. performed research;
S.S.S. contributed new reagents/analytic tools; J.G., S.S.S., and J.A.W. analyzed data; and
J.G., S.S.S., and J.A.W. wrote the paper.
The authors declare no conflict of interest.
1Present address: Banting and Best Department of Medical Research and the Donnelly
Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON,
Canada M5S 3E1.
2To whom correspondence should be addressed: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
activation of caspase-1. It has been suggested that in cells procaspase-1 exists
primarily as monomer. Upon binding to scaffolding proteins (NALPs, ASC,
etc.), procaspase-1 dimerizes and undergoes proteolytic activation. Mature
caspase-1 is in equilibrium between off- and on-conformations. Binding of
ligands at the active or allosteric site can shift the equilibrium toward the on-
or off-state. (B) Covalent labeling of apo-caspase-1. Irreversible active-site
inhibitors or allosteric compounds were used to trap caspase-1 into a stable
conformation for antibody selection.
Model and labeling design. (A) Proposed model for the dynamic
www.pnas.org?cgi?doi?10.1073?pnas.0812952106 PNAS ?
March 3, 2009 ?
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LC-MS (Waters). To prepare the off-form of caspase-1, a catalytic-inactive
caspase-1 Cys285Ala was incubated with 150 ?M of the allosteric inhibitor
[compound 34 or compound 11 (8)] at 4 °C overnight in the same labeling
buffer containing 1 mM ß-ME. For random biotinylation, the off-form of
caspase-1 was incubated with 15-fold excess sulfo-NHS-LC-biotin (Pierce) for
45 min at ambient temperature, and the reaction was stopped by buffer
exchange using a NAP-25 column (GE Healthcare).
Library Construction and Sorting. We modified the Fab-template phagemid
chain CDR-L3 to reduce wild-type Fab background. For the construction of
naïve libraries, the resulting phagemid was used as the ‘‘stop template’’ in a
mutagenesis reaction with oligonucleotides designed to repair simulta-
neously the stop codons and introduce designed mutations at the desired
sites, as described (16).
In sorting for on-form specific Fabs, the phage pool was cycled through
directly immobilized on 96-well Maxisorp plate (Thermo Fisher). Bound
phage were eluted with 100 mM HCl and neutralized with 1 M Tris, pH 8.0.
Phage were amplified in E. coli XL1-blue (Stratagene) with the addition of
M13-KO7 helper phage (New England Biolabs). In sorting for the off-form
specific Fabs, a solution-phase binding strategy was adapted for better
control over the selection and anti-selection process. The phage pool was
incubated for 2 h at room temperature with biotinylated allosteric con-
former before being captured on neutravidin or streptavidin (Pierce)
coated Maxisorp plates. The bound phage were then eluted and propa-
gated as described above. After selection, individual clones were picked
and grown in a 96-well deep well plate with 2YT broth supplemented with
carbenicillin and M13-KO7. The culture supernatants were used in phage
ELISAs to identify binding clones (33).
was converted into the Fab expression vector by deleting the sequence
encoding for the cP3 minor phage coat protein and inserting a ? terminator
sequence (GCTCGGTTGCCGCCGGGCGTTTTTTAT) downstream of the stop
codon at the end of CH1domain. Fab protein was secreted from E. coli 34B8
strain transformed with individual plasmids in low-phosphate medium at
30 °C for 26 h, as described (18). To generate IgG proteins, the variable
domains were subcloned into vectors designed for transient IgG expression in
CHO cells (18). Fab proteins were purified with protein A affinity chromatog-
Kinetic binding analyses were performed by surface plasmon resonance
(SPR) using a BIAcore 3000 (GE Healthcare). Ligand-bound or free caspase-1
dimers were immobilized on CM5 chips and serial dilutions of Fabs or IgGs
were injected. Binding responses on flow cells with immobilized caspase-1
cell. A 1:1 Languir model in BIAevaluation software (GE Healthcare) was used
for fitting the sensograms and the KDvalues were calculated from the ratios
Immunoprecipitation from THP-1 Cell Extracts. THP-1 cells were grown to a
Cells were lysed by Dounce homogenizer in ice-cold buffer (20 mM Hepes-
KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 0.1
by centrifugation at 500 ? g, 3,000 ? g, and 22,000 ? g for 10 min each.
Aliquots were incubated with or without IgGs overnight at 4 °C and immu-
nocomplexes were recovered by protein-G agarose beads. Presence of pro-
caspase-1 and caspase-1 were visualized by Western blot analysis.
Immunofluoresence Microscopy. THP-1 cells were grown to the density of 5 ?
105cell/ml and differentiated with 0.5 ?M PMA for 3 h and allowed to attach
to no. 11⁄2 glass coverslips overnight. Cells were treated with 1 mM LPS for 2 h
followed by 5 mM ATP for 30 min before fixation and mild detergent perme-
abilization. After blocking with 10% BSA for 1 h, IgGonwas added at 100 ?M
concentration for 1 h. After 3 washes with PBS ? 0.1% Triton X-100, the cells
were stained for 1 h with Alexa Fluor 488 conjugated goat anti-human IgG
antibody (Invitrogen). Cells were washed 3 times and mounted with ProLong
Gold containing DAPI (Invitrogen). Images were recorded on a Nikon 6D
High-Throughput Microscope equipped with a Photometrics Coolsnap HQ2
ACKNOWLEDGMENTS. We thank S. Birtalan, Y. Zhang, B. Li, G. Fuh, J. Scheer,
V. Chiang, and Y. Chen for advice and assistance. We also thank the Protein
Engineering Department and the sequencing, oligonucleotide synthesis, and
fermentation teams at Genentech for generous support, as well as K. Thorn
and the Nikon Imaging Center at University of California, San Francisco, for
help with immunofluorescence microscopy. This work was supported by Na-
tional Institutes of Health Grant 5R01AI070292–02 and the Sandler Family
Foundation gift (to J.A.W.).
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