4L39

Crystal structure of GH3.12 from Arabidopsis thaliana in complex with AMPCPP and salicylate

Summary for 4L39

• Entry DOI: 10.2210/pdb4l39/pdb
• Related: 4EPM 4EQ4 4EQL 4EWV
• Descriptor: 4-substituted benzoates-glutamate ligase GH3.12, MAGNESIUM ION, 2-HYDROXYBENZOIC ACID, ... (5 entities in total)
• Functional Keywords: acyl acid amido synthase, protein-ligand complex, magnesium, ligase
• Biological source: Arabidopsis thaliana (mouse-ear cress)
• Total number of polymer chains: 2
• Total formula weight: 132835.82

Authors

Zubieta, C.,Jez, J.M.,Brown, E.,Marcellin, R.,Kapp, U.,Round, A.,Westfall, C. (deposition date: 2013-06-05, release date: 2013-10-02, Last modification date: 2023-09-20)

Primary citation

Round, A.,Brown, E.,Marcellin, R.,Kapp, U.,Westfall, C.S.,Jez, J.M.,Zubieta, C.
Determination of the GH3.12 protein conformation through HPLC-integrated SAXS measurements combined with X-ray crystallography.
Acta Crystallogr.,Sect.D, 69:2072-2080, 2013

PubMed Abstract: 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.

PubMed: 24100325
DOI: 10.1107/S0907444913019276

Experimental method

X-RAY DIFFRACTION (2.81 Å)