期刊
JOURNAL OF MOLECULAR RECOGNITION
卷 32, 期 1, 页码 -出版社
WILEY
DOI: 10.1002/jmr.2755
关键词
crystal structure; nanobody; triclocarban; hapten; VHH
资金
- CSIC-UDELAR [Grupos 149]
- NIH-ORIP HEI [S10OD021527]
- National Institute of General Medical Sciences from the National Institutes of Health [P41 GM103403]
- US Department of Energy Office of Biological and Environmental Research
- Plexxikon Inc.
- National Science Foundation [1330685]
- National Institutes of Health [GM094625, GM082250, GM073210, GM105404]
- Sandler Foundation
- University of California Office of the President, Multicampus Research Programs and Initiatives [MR-15-328599]
- National Institutes of Health, National Institute of General Medical Sciences [P41GM103393]
- DOE Office of Biological and Environmental Research
- US Department of Energy, Office of Science, Office of Basic Energy Sciences [DE-AC02-76SF00515]
- U.S. Department of Agriculture [CA-D-MCB-7165-H]
- UDELAR [Grupos 149]
- M.D. Anderson Cancer Center
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1330685] Funding Source: National Science Foundation
The variable VHH domains of camelid single chain antibodies have been useful in numerous biotechnology applications due to their simplicity, biophysical properties, and abilities to bind to their cognate antigens with high affinities and specificity. Their interactions with proteins have been well-studied, but considerably less work has been done to characterize their ability to bind haptens. A high-resolution structural study of three nanobodies (T4, T9, and T10) which have been shown to bind triclocarban (TCC, 3-(4-chlorophenyl)-1-(3,4-dichlorophenyl)urea) with near-nanomolar affinity shows that binding occurs in a tunnel largely formed by CDR1 rather than a surface or lateral binding mode seen in other nanobody-hapten interactions. Additional significant interactions are formed with a non-hypervariable loop, sometimes dubbed CDR4. A comparison of apo and holo forms of T9 and T10 shows that the binding site undergoes little conformational change upon binding of TCC. Structures of three nanobody-TCC complexes demonstrated there was not a standard binding mode. T4 and T9 have a high degree of sequence identity and bind the hapten in a nearly identical manner, while the more divergent T10 binds TCC in a slightly displaced orientation with the urea moiety rotated approximately 180 degrees along the long axis of the molecule. In addition to methotrexate, this is the second report of haptens binding in a tunnel formed by CDR1, suggesting that compounds with similar hydrophobicity and shape could be recognized by nanobodies in analogous fashion. Structure-guided mutations failed to improve binding affinity for T4 and T9 underscoring the high degree of natural optimization.
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