Conserved polar residues stabilize transmembrane domains and promote oligomerization in human nucleoside triphosphate diphosphohydrolase 3

Abstract

Polar residues play essential roles in the functions of transmembrane helices by mediating and stabilizing their helical interactions. To investigate the structural and functional roles of the conserved polar residues in the N- and C-terminal transmembrane helices of human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) (N-terminus, S33, S39, T41, and Q44; C-terminus, T490, T495, and C501), each was singly mutated to alanine. The mutant proteins were analyzed for enzymatic activities, glycosylation status, expression level, and Triton X-100 detergent sensitivity. The Q44A mutation decreased Mg-ATPase activity by approximately 70% and abolished Triton X-100 detergent inhibition of Ca-dependent nucleotidase activities while greatly attenuating Triton X-100 inhibition of Mg-dependent nucleotidase activities. The polar residues were also mutated to cysteine, singly and in pairs, to allow a disulfide cross-linking strategy to map potential inter- and intramolecular hydrogen bond interactions. The results support the centrality of Q44 for the strong intermolecular interactions driving the association of the N-terminal helices of two NTPDase3 monomers in a dimer, and the possibility that T41 may play a role in the specificity of this interaction. In addition, S33 and C501 form an intramolecular association, while S39 and T495 may contribute to helical interactions involved in forming higher-order oligomers. Lastly, Tween 20 substantially and selectively increases NTPDase3 activity, mediated by the transmembrane helices containing the conserved polar residues. Taken together, the data suggest a model for putative hydrogen bond interactions of the conserved polar residues in the transmembrane domain of native, oligomeric NTPDase3. These interactions are important for proper protein expression, full enzymatic activity, and susceptibility to membrane perturbations.

Figure 1. Rationale for NTPDase3 mutations generated and analyzed in this study

Panel A Multiple sequence alignment of the N- and C-terminal TM helices in NTPDase3. The polar residues present in human NTPDase3 which were mutated are bolded and underlined. The sequence in GenBank for the cow NTPDase3 terminates prior to the C-terminal TM helix, and is therefore not known and not shown in the figure. Panel B. Helical wheel analysis of human NTPDase3 TM helices. The polar TM amino acids are represented by filled black circles. The helices are depicted as viewed from the extracellular side of the cell membrane, with the size of the circles (representing the amino acids) decreasing with increasing distance from the cell surface. Panel C. The potential TM polar residue hydrogen bond pairings. Based on positions on the helical wheel diagram Q44, S39, and S33 on the N-terminal TM helix are predicted to be on the same face and at the same depth as T490, T495, and C501 on the C-terminal TM helix (Panel B), respectively. Note that intra-molecular or inter-molecular (or both, in the case of Q44 which can form 2 hydrogen bonds) are possible for these polar residues present in the NTPDase3 dimer, which includes 2 N-terminal and 2 C-terminal TM helices.

Figure 2. Alanine and cysteine substitution at Q44 greatly attenuates the detergent inhibition of NTPDase3 enzymatic activities by Triton X-100

Panel A. Effect of alanine substitutions on the Triton X-100 treated enzyme activities. Panel B. Effect of cysteine substitutions on the Triton X-100 treated enzyme activities. The activities are expressed as the percent control activity measured in the absence of Triton X-100. Values represent the mean ± standard deviation from three separate experiments. The dashed horizontal line indicates no change of activity in the presence of Triton X-100.

Figure 3. Q44C NTPDase3 mutant form spontaneous, inter-molecular disulfide bond dimers

Panel A. Western blot analysis for the presence of spontaneous, inter-molecular disulfide bond formation. Panel B. Analysis of the Q44C inter-molecular disulfide linked dimer which is resistant to reduction by 30 mM DTT (Panel A). BME = boiled for 5 minutes in 2.86 M (20%) BME, DTT = boiled for 5 minutes in 200 mM DTT, NR = boiled for 5 minutes in the absence of reductant. The monomer (M) and dimer (D) bands are labeled. Bands migrating above the dimer are higher order oligomers.

Figure 4. Oxidative cross-linking and alkylation of NTPDase3 polar residue hydrogen bonding pair (double cysteine) mutants

Panel A. Lack of alkylation with MalPEG and lack of CuPhen-induced dimer formation suggests quantitative, intra-molecular cross-linking of the S33C/C501 mutant. Panel B. CuPhen-induced, inter-molecular interactions of the S39C/T495C mutant. Panel C. Spontaneous inter- and CuPhen-induced, intra-molecular interactions of the Q44C/T490C mutant. The monomer (M) and dimer (D) bands are indicated.

Figure 5. Model of the putative TMD hydrogen bond helical interactions in the NTPDase3 dimer

Panel A. Helical wheel model depicting the putative inter- and intra-molecular hydrogen bonding pattern of the conserved polar residues in the TMD of NTPDase3. Panel B. A 3-D model depicting the putative inter- and intra-molecular hydrogen bonding pattern of the conserved polar residues in the TMDs of NTPDase3. In the figure, the tops of the helical cylinders are at the interface between the cell membrane and the extracellular portion of the NTPDase containing the active site. Monomer 1, denoted as N1 (N-terminal helix) and C1 (C-terminal helix), is represented as solid cylinders with polar residues important for dimer interactions underlined and solid lines representing the hydrogen bond helical interactions. Monomer 2, with the TM helices denoted as N2 and C2, is represented as dotted cylinders with dotted lines representing the hydrogen bond helical interactions.

Figure 6. S39C and T495C may participate in hydrogen bond interactions involved in forming tetrameric NTPDase3

The mutants were oxidized with CuPhen and alkylated with MalPEG as described in the methods. Unlike the parent Q44C, a small amount of higher order oligomers are observed in the S39C/Q44C and T495C/Q44C double cysteine mutants. The monomer (M) and dimer (D) bands are labeled, and the positions of the higher order oligomers observed for Q44C/S39C and Q44C/T495C are indicated by asterisks (*).

Figure 7. The stimulatory effect of Tween 20 is specific for NTPDase3 and conserved among species

Panel A. Mg-ATPase assay of human, membrane-bound NTPDase isoenzymes in the presence of 0.1% Tween 20. Panel B. Mg-ATPase assay of NTPDase3 from three species in the presence of 0.1% Tween 20. The activities are expressed as the percent control activity measured in the absence of Tween 20. Values represent the mean ± standard deviation from three separate experiments. The dashed horizontal lines indicate no change of activity in the presence of Tween 20.

Figure 8. Tween 20 promotes oligomerization mediated by the TM helices of NTPDase3

Panel A. Effect of Tween 20 on glutaraldehyde (lysine specific) cross-linking efficiency via the non-transmembrane regions of C10S (wt-like) NTPDase3. Note less monomer remaining, and more oligomers formed, in the presence of Tween 20, in sharp contrast to the large attenuation of cross-linking observed with Triton X-100 detergent. Panel B. Effect of 0.1% Tween 20 or Triton X-100 on CuPhen cross-linking efficiency of the TM helices of the S39C/T495C double cysteine NTPDase3 mutant. Note the stark contrast in the inter-molecular cross-linking efficiencies obtained using Tween 20 versus Triton X-100 as the detergent.