期刊
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY
卷 69, 期 -, 页码 2072-2080出版社
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S0907444913019276
关键词
-
资金
- National Science Foundation [MCB-0904215]
- US Department of Agriculture National Institute of Food and Agriculture [MOW-2010-05240]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1157771] Funding Source: National Science Foundation
The combination of protein crystallography and small-angle X-ray scattering (SAXS) provides a powerful method to investigate changes in protein conformation. These complementary structural techniques were used to probe the solution structure of the apo and the ligand-bound forms of the Arabidopsis thaliana acyl acid-amido synthetase GH3.12. This enzyme is part of the extensive GH3 family and plays a critical role in the regulation of plant hormones through the formation of amino-acid-conjugated hormone products via an ATP-dependent reaction mechanism. The enzyme adopts two distinct C-terminal domain orientations with 'open' and 'closed' active sites. Previous studies suggested that ATP only binds in the open orientation. Here, the X-ray crystal structure of GH3.12 is presented in the closed conformation in complex with the nonhydrolysable ATP analogue AMPCPP and the substrate salicylate. Using on-line HPLC purification combined with SAXS measurements, the most likely apo and ATP-bound protein conformations in solution were determined. These studies demonstrate that the C-terminal domain is flexible in the apo form and favours the closed conformation upon ATP binding. In addition, these data illustrate the efficacy of on-line HPLC purification integrated into the SAXS sample-handling environment to reliably monitor small changes in protein conformation through the collection of aggregate-free and highly redundant data.
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