TY - JOUR
T1 - Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation
AU - Hattori, Takamitsu
AU - Lai, Darson
AU - Dementieva, Irina S.
AU - Montaño, Sherwin P.
AU - Kurosawa, Kohei
AU - Zheng, Yupeng
AU - Akin, Louesa R.
AU - ͆wist-Rosowska, Kalina M.
AU - Grzybowski, Adrian T.
AU - Koide, Akiko
AU - Krajewski, Krzysztof
AU - Strahl, Brian D.
AU - Kelleher, Neil L.
AU - Ruthenburg, Alexander J.
AU - Koide, Shohei
N1 - Funding Information:
We thank J. Osipiuk for assistance with data collection at the Advanced Photon Source, Drs. A. Gupta and S. Tanaka for assistance with X-ray structure determination, Dr. D. Kovar for access to a cell homogenizer, and Drs. A. Kossiakoff and M. Lugowski for access to cell culture equipment. This work was supported by National Institutes of Health (NIH) Grants R21 DA025725 and RC1 DA028779 (to S.K.) and GM067193 (to N.L.K.). B.D.S. acknowledges funding from the W. M. Keck Foundation. S.K., A.J.R., and N.L.K. acknowledge funding from the Chicago Biomedical Consortium, with support from the Searle Funds at the Chicago Community Trust. We acknowledge the use of the University of Chicago Genomics, Flow Cytometry, and Biophysics core facilities that are supported by the University of Chicago Comprehensive Cancer Center under NIH Grant P30 CA014599. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DEAC02-06CH11357.
PY - 2016/2/23
Y1 - 2016/2/23
N2 - Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This "antigen clasping" produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen- binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody-antigen recognition and suggests a strategy for developing extremely specific antibodies.
AB - Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. This "antigen clasping" produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen- binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody-antigen recognition and suggests a strategy for developing extremely specific antibodies.
KW - Antibody engineering
KW - Antibody validation
KW - Data reproducibility
KW - Epigenetics
KW - Protein-protein interaction
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U2 - 10.1073/pnas.1522691113
DO - 10.1073/pnas.1522691113
M3 - Article
C2 - 26862167
AN - SCOPUS:84959201050
VL - 113
SP - 2092
EP - 2097
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 8
ER -