Two-state selection of conformation-specific antibodies.
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, termed the on-form and the off-form. These conformation-trapped forms of the protease were used as antigens in alternating rounds of selection and antiselection for antibody antigen-binding fragments (Fabs) displayed on phage. After affinity maturation, 2 Fabs were isolated with K(D) values ranging from 2 to 5 nM, and each bound to their cognate conformer 20- to 500-fold more tightly than 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 general strategy for producing conformation-selective antibodies and show their utility for probing the distribution of caspase-1 conformational states in vitro and in cells.
Gao
Junjun
J
Department of Pharmaceutical Chemistry, University of California, 1700 4th Street, San Francisco, CA 94143, USA.
Sidhu
Sachdev S
SS
Wells
James A
JA
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D020170:Caspase 1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D019151:Peptide Library; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
D000906:Antibodies; D019151:Peptide Library; D020170:Caspase 1