Proline residues link the active site to transmembrane domain movements in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3)

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.

Keywords: Conserved proline residues; Ecto-nucleotidase; Linker region; NTPDase3; Site-directed mutagenesis; Transmembrane cross-linking.

Fig. 1

Rationale and experimental approach. a Multiple sequence alignments for portions of NTPDase sequences between the N- and C-terminal transmembrane (TM) helices and the extracellular domain containing the active site. Conserved proline residues found in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) are bolded and numbered, and the corresponding prolines in other NTPDases are bolded. The cysteine substitutions of rat NTPDase1/CD39 made in a previous study (A34C and S481C, [23]) are bolded and underlined, and guided the choice of the cysteine substitutions used in this work (V42C and G489C), which are bolded and underlined in the human NTPase3 sequence (top line of each alignment). To give a perspective as to the location of these conserved proline residues in the overall structure of NTPDase3, the locations of the end of the N-terminal TM helix, the beginning of the ACR1 region (N-terminal), the end of ACR5 region (C-terminal), and the beginning of the C-terminal TM helix are indicated by lines and italic labeling under the alignments. b A flow chart of the experimental approach utilized

Fig. 2

Adenosine triphosphate (ATP) attenuates the copper phenanthroline (CuPhen)-induced decrease in nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) Ca-ATPase activity. Nucleotidase activities were assayed as described in “Materials and methods.” Values represent the mean percent activity relative to the control samples ± standard deviation from three separate experiments

Fig. 3

Adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine 5’-(β,γ-imido)triphosphate (AMP-PNP) binding at the extracellular domain decreases the copper phenanthroline (CuPhen) cross-linking efficiency of V42C and G489C “sensor” background nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) mutants. CuPhen cross-linking was performed, and the amount of monomer remaining as compared to the untreated control was quantified as described in “Materials and methods.” a V42C NTPDase3 mutant results. b G489C NTPDase3 mutant results. Above each mutant percent monomer data bar is a representative Western blot. The bar graph on the bottom of each panel is the quantification of three such Western experiments, expressed as the mean percent of untreated monomer remaining ± standard deviation. Asterisks represent statistical significance (p ≤ 0.05) between mutant proteins oxidized with CuPhen in the presence versus the absence of nucleotide or Pi

Fig. 4

Copper phenanthroline (CuPhen) cross-linking efficiency of proline to alanine mutants in the presence and absence of 5 mM adenosine triphosphate (ATP). Cross-linking was performed, and ATP was added before 5 mM CuPhen, as described in “Materials and methods.” a V42C proline to alanine mutants. b G489C proline to alanine mutants

Fig. 5

Specific proline to alanine substitutions abolish the adenosine triphosphate (ATP)-induced decrease in copper phenanthroline (CuPhen) cross-linking efficiency. Cross-linking was performed, and the efficiency of cross-linking was quantified by comparing the amount of monomer remaining to the monomer in the untreated control, as described in “Materials and methods.” a V42C proline to alanine mutants. b G489C proline to alanine mutants. Values represent the mean percent of monomer remaining after CuPhen ± standard deviation from three separate experiments (a single representative Western blot experiment for each mutant is shown in Fig. 4). Asterisks represent statistically significant differences between each proline to alanine mutant in the presence versus the absence of ATP (p ≤ 0.05). The boxes around the mutations used to label the x-axis denote mutants that abolish the ATP-induced decrease in CuPhen cross-linking. The P485A mutation in the G489C background (b) abolishes transmembrane CuPhen cross-linking both in the absence and presence of ATP and is annotated by a strikethrough ellipse

Fig. 6

Summary of the proline to alanine nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) mutant data. Shown are cartoon representations of two monomers of human NTPDase3. On the left, the results for the proline substitutions in the V42C N-terminal transmembrane (TM) cysteine substitution background are schematically represented. On the right, the results for the proline substitutions in the G489C C-terminal TM cysteine substitution background are schematically represented. The N- and C-terminal TM helices are represented by gray cylinders, the cell membrane by thin horizontal lines, and the “linker regions” containing the conserved and mutated proline residues are indicated by straight lines connecting the TM helices with ACR1 and ACR5 regions (indicated by ellipses). The majority of the extracellular portion of NTPDase3, consisting of two lobes, is represented by a free-form curve, and forms the active site crevice, shown with substrate (adenosine triphosphate (ATP)) present. Proline to alanine mutations which decrease nucleotidase activities (normalized for expression levels) are indicated by bold type. Those mutations which abolish the ability of substrate (ATP) to diminish the cross-linking efficiency of the introduced TM helix cysteine residues (“uncoupling mutations”) are indicated by boxes drawn around the mutations. The P485A mutation in the G489C background is fully active but abolishes TM copper phenanthroline cross-linking both in the absence and presence of ATP and is annotated by a strikethrough ellipse