Abstract
The active sites of the membrane-bound nucleoside triphosphate diphosphohydrolases (NTPDases) regulate and are regulated by coordinated and spatially distant movements of their transmembrane helices, modulating enzyme activity, and substrate specificity. Using site-directed mutagenesis, the roles of the conserved proline residues (N-terminal: P52 and P53; C-terminal: P472, P476, P481, P484, and P485) of human NTPDase3, located in the “linker regions” that connect the N- and C-terminal transmembrane helices with the extracellular active site, were examined. Single cysteine substitutions were strategically placed in the transmembrane domain (N-terminal helix: V42C; C-terminal helix: G489C) to serve as cross-linking “sensors” of helical interactions. These “sensor” background mutant proteins (V42C and G489C NTPDase3) are enzymatically active and are cross-linked by copper phenanthroline less efficiently in the presence of adenosine triphosphate (ATP). Proline to alanine substitutions at P53, P481, P484, and P485 in the V42C background, as well as P53, P481, and P484 in the G489C background, exhibited decreased nucleotidase activities. More importantly, alanine substitutions at P53 and P481 in the V42C background and P481 in the G489C background no longer exhibited the ATP-induced decrease in transmembrane cross-linking efficiency. Interestingly, the P485A mutation abolished oxidative cross-linking at G489C both in the presence and absence of ATP. Taken together, these results suggest a role for proline residues 53 and 481 in the linker regions of human NTPDase3 for coupling nucleotide binding at the enzyme active site to movements and/or rearrangements of the transmembrane helices necessary for optimal nucleotide hydrolysis.
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Abbreviations
- NTPDase3:
-
Nucleoside triphosphate diphosphohydrolase 3
- TM:
-
Transmembrane
- TMD:
-
Transmembrane domain
- CuPhen:
-
Copper phenanthroline
- NEM:
-
N-ethylmaleimide
- MOPS:
-
3-(N-Morpholino)-propanesulfonic acid
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This work was supported by the National Institutes of Health R01 grant HL72382/HL72382-S1 to T.L.K.
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Gaddie, K.J., Kirley, T.L. Proline residues link the active site to transmembrane domain movements in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3). Purinergic Signalling 6, 327–337 (2010). https://doi.org/10.1007/s11302-010-9180-0
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DOI: https://doi.org/10.1007/s11302-010-9180-0