4A5A: Crystal Structure Of The C258s/c268s Variant Of Toxoplasma Gondii Nucleoside Triphosphate Diphosphohydrolase 3 (ntpdase3) In Complex With Magnesium And Amppnp

Citation:
Abstract
The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 A, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258-268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys(258)-Cys(268) show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12 degrees domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg(2+) to a resolution of 2.85 A indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg(492) and Glu(493) in NTPDase1 by glycines in NTPDase3.
PDB ID: 4A5ADownload
MMDB ID: 95466
PDB Deposition Date: 2011/10/24
Updated in MMDB: 2013/12
Experimental Method:
x-ray diffraction
Resolution: 2.85  Å
Source Organism:
Similar Structures:
Biological Unit for 4A5A: tetrameric; determined by author and by software (PISA)
Molecular Components in 4A5A
Label Count Molecule
Proteins (4 molecules)
4
Nucleoside-triphosphatase 1
Molecule annotation
Chemicals (8 molecules)
1
4
2
4
* Click molecule labels to explore molecular sequence information.

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