The proposed channel-enzyme transient receptor potential melastatin 2 does not possess ADP ribose hydrolase activity
Transient receptor potential melastatin 2 ( TRPM2 ) belongs to the TRP protein family and is predominantly expressed in brain neurons , spleen , bone marrow , leukocytes , phagocytes , pancreatic β cells , and cardiomyocytes ( Nagamine et al . , 1998; Perraud et al . , 2001; Togashi et al . , 2006 ) . It forms Ca2+-permeable nonselective cation channels which are activated under oxidative stress , and contribute to the Ca2+ signals that trigger cell migration and chemokine production in immune cells ( Yamamoto et al . , 2008 ) and insulin secretion in the pancreas ( Uchida et al . , 2011 ) . Under pathological conditions , such as post-ischemic reperfusion injury ( Hara et al . , 2002; Kaneko et al . , 2006 ) and potentially in Alzheimer's disease ( Fonfria et al . , 2005 ) , TRPM2 activity leads to Ca2+ dysregulation and cell death . On the other hand , reduced activity of TRPM2 might be linked to amyotrophic lateral sclerosis and Parkinson’s disease dementia ( Hermosura et al . , 2008 ) , as well as bipolar disorder ( McQuillin et al . , 2006 ) . TRPM2 forms homotetramers with a transmembrane architecture similar to that of voltage-gated cation channels ( Figure 1A ) . In addition , it contains a large cytosolic N-terminal domain of unknown function specific to the TRPM subfamily , and a C-terminal coiled-coil region ( CCR , Figure 1A ) followed by a small cytosolic domain ( NUDT9H ) . The NUDT9H domain exhibits a high degree of sequence similarity ( ~35% identity , ~45% similarity ) to the soluble , Mg2+-dependent mitochondrial pyrophosphatase NUDT9 ( Shen et al . , 2003 ) , which catalyzes the hydrolysis of adenosine 5'-diphosphoribose ( ADPR ) into AMP and ribose 5-phosphate ( R5P ) ( Figure 1B and C ) . TRPM2 is activated by co-application of cytosolic ADPR and Ca2+ ( Perraud et al . , 2001; Sano et al . , 2001; Csanády and Törocsik , 2009 ) , and the ADPR ligand was indeed shown to bind to the NUDT9H domain of the channel ( Grubisha et al . , 2006 ) . Based on its crystal structure , the mitochondrial enzyme is composed of two subdomains , termed Cap and Core ( Figure 1C , blue and brown ) . The Cap subdomain facilitates binding of the substrate ADPR , whereas the Core subdomain binds the substrate and catalyses its cleavage to AMP and R5P ( Shen et al . , 2003; Perraud et al . , 2003 ) . The Core subdomain harbors the Nudix ( nucleoside diphosphate linked moiety X ) box - a conserved motif ( Bessman et al . , 1996 ) responsible for recognition and hydrolysis of ADPR , present in a variety of phosphohydrolases ( Gabelli et al . , 2001; Kang et al . , 2003; Gabelli et al . , 2007; Huang et al . , 2008; Messing et al . , 2009; Boto et al . , 2011 ) including both NUDT9 and NUDT9H ( Perraud et al . , 2001 ) . 10 . 7554/eLife . 17600 . 003Figure 1 . The ligand-binding NUDT9H domain in the context of TRPM2 . ( A ) Topology map of a TRPM2 monomer , showing the N-terminal TRPM homology region ( dark blue ) , the six transmembrane helices which form the pore domain ( red and blue ) , and a C-terminal short coiled-coil region ( CCR , yellow ) followed by the NUDT9H domain ( gray ) which is homologous to the soluble mitochondrial ADPRase NUDT9 . ( B ) Hydrolysis of ADPR to AMP and R5P: reaction catalyzed by mitochondrial NUDT9 and reportedly also by the TRPM2 NUDT9H domain . ( C ) Crystal structure of mitochondrial NUDT9 ( PDB ID: 1QVJ ) illustrating Cap ( blue ) and Core ( brown ) subdomains of the protein , with the hydrolysis product R5P ( yellow ) and two Mg2+ ions ( magenta ) bound . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 003 The strong similarity between NUDT9 and NUDT9H raised the intriguing possibility that NUDT9H itself might act as an enzyme with ADPR hydrolase ( ADPRase ) activity . Indeed , previous work reported weak enzymatic activity for isolated NUDT9H ( Perraud et al . , 2001 , 2003 ) , compatible with the slow timescale of channel gating ( Csanády and Törocsik , 2009 ) , classifying TRPM2 among channel-enzymes ( 'chanzymes' ) . This small group of proteins , in which a single polypeptide chain forms both a transmembrane ion pore and a catalytically active domain , includes two other members of the TRPM subfamily ( TRPM6 and TRPM7 ) , and the Cystic Fibrosis Transmembrane Conductance Regulator ( CFTR ) chloride ion channel . In CFTR catalytic ( ATPase ) activity is strictly coupled to channel gating , which therefore follows a nonequilibrium cyclic mechanism ( Csanády et al . , 2010 ) . In TRPM7 catalytic ( phosphotransferase ) activity is not linked to pore gating ( Matsushita et al . , 2005 ) , but instead serves to regulate expression of TRPM7-dependent genes ( Krapivinsky et al . , 2014 ) . In the case of TRPM2 the physiological role of the proposed ADPRase activity has not yet been clarified , but we have previously shown that any hydrolytic activity of NUDT9H is not coupled to gating of the TRPM2 channel itself ( Tóth et al . , 2014 ) . Furthermore , our observation of intrinsic instability of several pyridine dinucleotides at basic pH ( Tóth et al . , 2015 ) raised the possibility of a similar instability for ADPR , which might lead to false positive results when assessing the hydrolytic activity of a putative slow ADPRase enzyme . It thus remained a question whether the NUDT9H domain truly exhibits any enzymatic activity , not coupled to TRPM2 gating . One way to address this would be to further analyze the TRPM2 ADPR-binding domain in isolation . However , in our hands NUDT9H proved to be extremely insoluble ( Tóth et al . , 2014 ) , despite its high degree of sequence similarity to highly soluble NUDT9 . For this reason we first constructed a series of chimeric proteins built from complementary polypeptide fragments of the two homologous proteins , and correlated solubility with NUDT9H sequence content . This approach allowed us to pinpoint the NUDT9H segments responsible for its insolubility , and to purify and investigate soluble chimeric proteins which contain up to ~90% of the native NUDT9H sequence . We then investigated enzymatic activities of such soluble chimeras in isolation , as well as their capacities to substitute for native NUDT9H in conferring ADPR-dependent gating to the full-length TRPM2 channel .
In a previous attempt to purify and biochemically characterize the TRPM2 NUDT9H domain in isolation we found it to be essentially insoluble , unlike its mitochondrial homologue NUDT9 ( Tóth et al . , 2014 ) . Because of this unexpected difference we further investigated the two proteins in an attempt to obtain a soluble analogue of NUDT9H . Prompted by the strong similarity between them ( Figure 2 ) , we generated chimeric constructs by swapping homologous segments between NUDT9 and NUDT9H . The crystal structure of mitochondrial NUDT9 ( [Shen et al . , 2003]; PDB ID: 1Q33 ) identified two subdomains - a 'Cap' and a 'Core' ( Figure 1C , blue and brown ) . Assuming structural similarity between NUDT9 and NUDT9H , these two structurally distinct regions of NUDT9 provided the basis for generating the first chimeric constructs ( Chi1/1A and Chi2/2A ) , obtained by exchanging the Cap and Core segments of the two native proteins ( Figure 3A ) . The NUDT9-Cap/NUDT9H-Core chimeras Chi1 and Chi1A were insoluble , whereas the NUDT9H-Cap/NUDT9-Core chimeras Chi2 and Chi2A were soluble , and Chi2 could be purified by Ni-affinity chromatography ( Figure 3—figure supplement 1 ) . The low solubility of NUDT9H is thus caused at least in part by its Core subdomain . 10 . 7554/eLife . 17600 . 004Figure 2 . Alignment of NUDT9H and NUDT9 sequences . Aligned ( Corpet , 1988 ) sequences of NUDT9H and NUDT9 , highlighting identical ( red ) and similar ( blue ) residues . The approximate border between Cap and Core subdomains of the proteins is indicated by two black arrows . Black line identifies the Nudix box , asterisks denote residues critical for the catalytic activity of NUDT9 . Green arrows and labels indicate transition points for sequence swapping in each chimera ( see also Figure 3 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 00410 . 7554/eLife . 17600 . 005Figure 3 . Representation of the various recombinant proteins used in this study . ( A ) Aligned block diagrams of recombinant constructs with segment lengths realistically scaled to reflect their actual sizes ( numbers of residues ) . The Cap and Core segments of NUDT9 ( blue ) and NUDT9H ( orange ) are separated by a vertical black line . Gaps emerging from the alignment of the two sequences ( Figure 2 ) are presented as thick lines with the respective color . The 15 amino-acid stretch from the upstream cytosolic linker sequence of TRPM2 ( gray ) , present in some chimeras , contains a native BlpI-site which allows subcloning the domain into full-length TRPM2 . Some constructs contain a C-terminal hexahistidine tag ( green ) or Twin-Strep tag ( magenta ) . Nudix boxes within Core segments are indicated as black lines ( see also Table 1 ) . ( B ) NUDT9 fragments replaced in the chimeras by NUDT9H sequence , mapped onto surface representation of NUDT9 crystal structure ( PDB ID: 1Q33 ) . Coloring highlights progressive replacement of NUDT9 sequence with NUDT9H sequence in chimeric constructs . The Cap subdomain ( light brown ) is replaced in Chi2 and Chi2A . A fragment from the Core ( orange ) , containing the Nudix box ( red ) , is also replaced in Chi3S . Additional Core fragment ( dark brown ) further extends the NUDT9H sequence in Chi4S , leaving only 36 C-terminal amino acids from NUDT9 ( blue ) . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 00510 . 7554/eLife . 17600 . 006Figure 3—figure supplement 1 . Purified recombinant proteins . Coomassie-stained SDS PAGE gels of the five purified recombinant proteins ( mitochondrial NUDT9 , three soluble chimeric constructs , and the NUDT9H domain of TRPM2 ) , as indicated above each gel slice . Molecular weights for marker ladder bands ( Precision , Bio-Rad ) are labeled in kDa . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 006 The soluble Chi2 contains only ~41% of NUDT9H sequence ( Table 1 ) and , notably , the catalytically important Nudix box in Chi2 originates from mitochondrial NUDT9 . To obtain soluble constructs which more closely resemble NUDT9H , the point of transition from NUDT9H- to NUDT9-sequence was advanced further downstream . For the next construct , Chi3 ( Figure 3A ) , this switch point was propagated about halfway through the Core segment , immediately after a well aligned , conserved stretch of amino acids ( Figure 2 ) which forms a reentrant loop structure , pivotal for anchoring the Cap to the Core segment in NUDT9 . A small fraction of this Chi3 chimera , which contains ~77% of NUDT9H sequence ( Table 1 ) , including its Nudix box , remained soluble and could be purified . We then advanced the transition point further downstream , about halfway through the remainder of the NUDT9 sequence in Chi3 . This new construct ( Chi4 ) is formed by ~91% of NUDT9H sequence ( Figure 2 , Figure 3A and B ) , but a small amount of it still remained in the soluble fraction which allowed us to purify it . The low yields of soluble Chi3 and Chi4 could be robustly increased using a slower induction protocol ( 0 . 1 mM IPTG , 25°C , overnight ) ; and replacement of the hexahistidine tag with a Twin-Strep tag ( Chi3S and Chi4S ) greatly improved protein purity , as confirmed by SDS-PAGE ( Figure 3—figure supplement 1 ) and mass spectrometry analysis of the purified Chi4S sample . Indeed , using this slow induction protocol even some soluble NUDT9H protein could be obtained and purified ( Figure 3—figure supplement 1 , right ) ; however , this preparation precipitated overnight at 4°C . Thus , Chi4S remained the best NUDT9H model that we were able to obtain in stable , soluble form ( Table 1 ) . 10 . 7554/eLife . 17600 . 007Table 1 . Properties of the recombinant proteins . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 007ConstructM . W . ( kDa ) Theor . pI*% NUDT9H†ecOpt‡Nudix§SolubleEnzymeNUDT934 . 06 . 880YesNUDT9YesYesNUDT9H31 . 58 . 34100YesNUDT9HNo¶NoChi131 . 79 . 3059YesNUDT9HNoN/A††Chi1A32 . 39 . 1659NoNUDT9HNoN/AChi235 . 76 . 2741NoNUDT9YesYesChi2A34 . 65 . 9141NoNUDT9YesN/AChi3S35 . 46 . 4077YesNUDT9HYes ( 25°C ) **NoChi4S35 . 87 . 1691YesNUDT9HYes ( 25°C ) **No* - Theoretical pI of the construct ( Protein Calculator v3 . 4 ) . † - Percentage of NUDT9H sequence ( TRPM2 residues 1236-1503 ) contained in the construct . ( The segment in brackets does not include the 15 amino-acid upstream stretch from TRPM2 ) . ‡ - Gene sequence optimized for expression in E . coli . ‘No’ denotes that the original , human nucleotide sequence ( Nagamine et al . , 1998 ) was used . § - Denotes the origin ( NUDT9 or NUDT9H ) of the Nudix box sequence in the construct . ¶ - Small fraction of the protein is soluble when expressed at 25°C . ** - Small fraction of the protein is soluble when expressed at 37°C , reasonably soluble when expressed at 25°C . †† - Not Available The solubility and relative stability of mitochondrial NUDT9 and three of our chimeric constructs ( Chi2 , 3S , 4S ) allowed purification of milligram amounts of these recombinant proteins to reasonable homogeneity ( Figure 3—figure supplement 1 ) and assessment of their enzymatic activities . Furthermore , despite its long-term instability , we also subjected freshly purified NUDT9H ( Figure 3—figure supplement 1 , right ) to enzyme activity assays . Following a 2-hr incubation of 1 μM of each protein with 10 mM ADPR substrate and 16 mM Mg2+ ( Perraud et al . , 2003 ) , TLC analysis of the reaction mixtures confirmed that NUDT9 and Chi2 ( which contain the NUDT9-type Nudix box ) are both active ADPRases , with turnover rates sufficient to cause complete – or near-complete – conversion of ADPR into AMP and R5P within this time frame ( Figure 4A and B; R5P is invisible on the TLC ) . In contrast , Chi3S , 4S and NUDT9H ( which contain the NUDT9H-type Nudix box , Figure 3A ) all failed to hydrolyze the nucleotide under the same conditions ( Figure 4C–E ) : this same overall pattern was observed for all six reaction conditions tested , which included three different pH values ( 6 . 5 , 7 . 1 and 8 . 5 ) in the absence or presence of 100 mM NaCl ( Figure 4A–E , reaction buffers 1–6 ) . 10 . 7554/eLife . 17600 . 008Figure 4 . Enzymatic activity of NUDT9 , NUDT9H , and chimeras . ( A–E ) 10 mM ADPR ( lane C1 ) and 10 mM AMP ( lane C2 ) controls in water . Lanes 1–6 show reaction mixtures of 1 μM purified NUDT9 ( A ) , Chi2 ( B ) , Chi3S ( C ) , Chi4S ( D ) , or NUDT9H ( E ) , incubated with 10 mM ADPR and 16 mM MgCl2 in 50 mM RB1-6 , respectively , for 2 hr at room temperature . ( F ) Top slice: TLC-detection limit for AMP: 0 . 1–0 . 9 mM water-dissolved AMP in 0 . 1-mM incrementing steps . Middle slice: ADPR self-degradation . The slice shows only the AMP position of ( from left to right ) : 10 mM ADPR control ( in water ) , 1 mM AMP control ( in water ) , 10 mM AMP control ( in water ) , and 1 , 2 , 5 , 10 , 15 and 20 mM ADPR incubated for 24 hr at room temperature in RB3 ( pH 8 . 5 ) ( last six lanes ) . Bottom slice: ADPR self-degradation in the absence and presence of Chi4S , showing only the AMP position . From left to right: 10 mM ADPR control ( in water ) ; 10 mM AMP control ( in water ) ; 10 mM ADPR in RB3 ( pH 8 . 5 ) ; 10 mM ADPR in RB6 ( pH 8 . 5/NaCl ) ; 0 . 1 μM Chi4S + 10 mM ADPR in RB3; 0 . 1 μM Chi4S + 10 mM ADPR in RB6; 1 μM Chi4S + 10 mM ADPR in RB3; 1 μM Chi4S + 10 mM ADPR in RB6 . Except for the controls in lanes 1–2 , all samples also contained 16 mM MgCl2 , and were incubated for 24 hr at room temperature . In all Panels 1-μl aliquots were loaded for each sample . The three slices of Panel F can be seen as full-scale TLC plates in Figure 4—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 00810 . 7554/eLife . 17600 . 009Figure 4—figure supplement 1 . Full-scale TLC images of the slices shown in Panel F on Figure 4 . ( A ) Image of entire TLC plate from which the top slice in Figure 4F was excised: TLC-detection limit for AMP . Lanes 1–9 show 0 . 1–0 . 9 mM water-dissolved AMP , respectively , in 0 . 1-mM incrementing steps . ( B ) Image of entire TLC plate from which the middle slice in Figure 4F was excised: ADPR self-degradation . Lane 1: 10 mM ADPR control ( in water ) ; lane 2: 1 mM AMP control ( in water ) ; lane 3: 10 mM AMP control ( in water ) ; lanes 4–9: 1 , 2 , 5 , 10 , 15 and 20 mM ADPR , respectively , incubated for 24 hr at room temperature in RB3 ( pH 8 . 5 ) . ( C ) Image of entire TLC plate from which the bottom slice in Figure 4F was excised . ADPR self-degradation in the absence and presence of Chi4S . Lane 1: 10 mM ADPR control ( in water ) ; lane 2: 10 mM AMP control ( in water ) ; lane 3: 10 mM ADPR in RB3 ( pH 8 . 5 ) ; lane 4: 10 mM ADPR in RB6 ( pH 8 . 5/NaCl ) ; lane 5: 0 . 1 µM Chi4S + 10 mM ADPR in RB3; lane 6: 0 . 1 µM Chi4S + 10 mM ADPR in RB6; lane 7: 1 µM Chi4S + 10 mM ADPR in RB3; lane 8: 1 µM Chi4S + 10 mM ADPR in RB6 . Except for the controls in lanes 1–2 , all samples also contained 16 mM MgCl2 , and were incubated for 24 hr at room temperature . In all Panels 1-μl aliquots were loaded for each sample . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 00910 . 7554/eLife . 17600 . 010Figure 4—figure supplement 2 . Spontaneous ADPR degradation at basic pH , monitored with TLC . The six TLC slices show only the AMP position of 10 mM ADPR control in water ( lane 1 ) , 1 mM AMP control in water ( lane 2 ) , 10 mM AMP control in water ( lane 3 ) , and 1 , 2 , 5 , 10 , 15 and 20 mM ADPR , respectively , in RB3 ( pH 8 . 5 ) ( lanes 4–9 ) . The ADPR samples were incubated at room temperature for different time durations , as indicated to the right of each slice . In all slices 1-μl aliquots were loaded for each sample . The 24h-slice is the same experiment shown in Figure 4F , middle slice ( and in Figure 4—figure supplement 1B ) . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 010 The reported enzymatic activity of NUDT9H is about 100-fold lower than that of NUDT9 ( Perraud et al . , 2001 , 2003 ) . Closer inspection of the TLC plates of the inactive proteins indeed revealed very weak AMP spots appearing in the reaction mixtures at basic pH ( Figure 4C–E , lanes 3 and 6 ) . The low sensitivity of the TLC technique requires the presence of at least 0 . 2–0 . 3 mM AMP for visual detection ( Figure 4F , top slice ) . Thus , assuming 2–300 μM AMP produced by 1 μM protein within our 2-hr incubation would suggest apparent kcat values of 0 . 03–0 . 04 s−1 for Chi3S , 4S and NUDT9H at pH 8 . 5 and 25°C , in good agreement with the previous reports on NUDT9H ( kcat ~0 . 06 s−1 at pH 9 and 37°C ) . However , our prior observation of spontaneous degradation of several dinucleotides at basic pH ( Tóth et al . , 2015 ) prompted us to consider inherent instability of ADPR at basic pH as a possible reason for the observed slow AMP accumulation . Indeed , we found that ADPR undergoes slow degradation to AMP and R5P at pH 8 . 5 ( Figure 4F , middle slice; also see Figure 4—figure supplement 2 ) , highlighting that care must be taken to distinguish spontaneous hydrolysis from true enzymatic activity . At 25°C and pH 8 . 5 ~20% of ADPR degraded spontaneously in 24 hr ( Figure 4F , middle slice , compare the two lanes marked by asterisks ) , indicating a decay time constant of ~100 h , and thus ~2% degradation in 2 hr: this rate of spontaneous degradation indeed fully accounts for the weak AMP spots observed for Chi3S , 4S , and NUDT9H at basic pH . More importantly , addition of either 0 . 1 or 1 μM Chi4S to ADPR-containing reaction mixtures at basic pH , either with ( RB6 ) or without ( RB3 ) 100 mM NaCl , did not affect the rate of spontaneous AMP accumulation ( Figure 4F , bottom slice ) . This clearly demonstrates that Chi4S does not possess any hydrolytic activity towards ADPR , most likely due to critical substitutions in its Nudix box ( Figure 2; cf . , [Perraud et al . , 2003] ) , and that the observed slow accumulation of AMP is merely caused by ADPR instability at basic pH . We noted slower enzymatic activity for Chi2 at pH 6 . 5 and 7 . 1 compared to 8 . 5 ( Figure 4B ) , whereas ADPR hydrolysis by NUDT9 appeared complete at all pH values ( Figure 4A ) . To test whether such pH dependence might also apply for NUDT9 , but could not be observed at the chosen timescale of 2 hr , we analyzed for both enzymes the time course of AMP accumulation in ADPR-saturated reaction mixtures at the three different pH values ( in RB1 , RB2 and RB3 ) . Both NUDT9 and Chi2 showed a clear preference for basic pH , causing AMP accumulation to be fastest at pH 8 . 5 and slowest at pH 6 . 5 ( Figure 5A and B ) . The calculated kcat values ( in s−1 ) quantify this pH dependence of catalytic rates for both proteins ( Figure 5C ) , and reveal higher sensitivity for Chi2 ( ~5 . 8-fold decrease from pH 8 . 5 to 6 . 5 ) compared to NUDT9 ( ~1 . 4-fold decrease from pH 8 . 5 to 6 . 5 ) . Spontaneous degradation of ADPR at basic pH ( Figure 4F , middle slice ) is not a major contributor to this pH dependence , because the total duration of these kinetic experiments ( ~20 min ) was too short for that effect to become significant: correction for the ~30 μM AMP which forms spontaneously in 20 min at pH 8 . 5 would amount to a downward adjustment of those kcat values ( Figure 5C , black bars ) by only ~0 . 05 s−1 . Furthermore , whereas we cannot exclude the possibility that the two proteins were applied at slightly different concentrations , and that this may have caused the observed differences in their absolute reaction rates , investigating pH dependence of activity of a given enzyme ( i . e . the same protein stock ) is not subject to such limitations . Of note , the kcat of 9 s−1 for NUDT9 at pH 8 . 5 is very similar to previously reported values ( ~8 s−1 , [Perraud et al . , 2001 , 2003] ) . 10 . 7554/eLife . 17600 . 011Figure 5 . Enzyme kinetics of Chi2 and NUDT9 . ( A ) Representative TLC images showing the time courses ( in minutes ) of hydrolysis of ADPR ( 10 mM ) by 0 . 5 μM Chi2 ( top ) or NUDT9 ( bottom ) at 3 different pH values ( RB1 , RB2 and RB3 ) . The slices show only the position of the AMP spots . Control 1 ( C1 ) : 5 mM AMP in water . Control 2 ( C2 ) : 10 mM AMP in water . ( B ) AMP accumulation during ADPR hydrolysis by Chi2 ( circles ) and NUDT9 ( triangles ) at 3 different pH values . Densities of AMP spots ( such as those shown in Panel A ) were normalized to the density of the control AMP spot ( 5 mM ) on the same TLC sheet . For each time point mean ± SEM from 3 experiments is shown . Straight lines are linear regression fits to the averaged data points . ( C ) Estimates of kcat ( s−1 ) for both proteins at each pH , calculated from the fitted slopes in B ( mean ± SEM; mean values are also printed above each bar ) . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 011 The TLC assay allows unambiguous identification of the reaction product AMP based on its chromatographic mobility , but its resolution is limited . As an independent approach , we therefore verified enzymatic activities at both 37°C ( Figure 6A ) and room temperature ( RT , Figure 6B ) for NUDT9 , Chi4S , and NUDT9H using the more sensitive colorimetric detection of inorganic phosphate ( Pi ) , liberated from both ADPRase products ( AMP and R5P ) by co-applied alkaline phosphatase ( AP ) ( Rafty et al . , 2002 ) . As a validation of this coupled assay , no significant amounts of contaminating Pi were found in either the applied stock of AP ( 5–6 U ) , or in 1-mM solutions of ADPR , AMP , or R5P ( Figure 6A–B , first four group of bars ) . Moreover , in the 30-min time frame of our incubation AP released stoichiometric amounts ( ~1 mM ) of Pi from either AMP or R5P ( but not from ADPR ) , confirming that under our experimental conditions the AP reaction is not rate limiting for the coupled assay ( Figure 6A–B , groups 5–7 ) , releasing 2 mM of Pi per mM ADPR hydrolyzed . When NUDT9 ( 5 or 50 nM ) was co-applied with 1 mM ADPR and AP , the released Pi roughly scaled with the applied amount of protein , signalling specific ADPR hydrolysis by NUDT9 ( Figure 6A–B , groups 8–9 ) . Comparison of released [Pi] for assays performed at pH values between 6 . 5 and 9 . 0 confirmed modest but significant enhancement of NUDT9 activity at basic pH ( Figure 6A–B , compare four colored bars within groups 8–9 ) . Molecular turnover rates ( kcat ) , calculated from the 5-nM NUDT9 data , were in the range of ~4–11 s−1 ( Figure 6D ) , in good agreement with the estimates from the TLC assay ( Figure 5C ) . In contrast , co-applying 5 or 50 nM of either Chi4S or NUDT9H with 1 mM ADPR and AP did not result in any significant Pi release ( Figure 6A–B , groups 10–13; expanded in Figure 6C ) : the small concentrations of Pi released in these assays ( corresponding to the hydrolysis of ~1% of the applied 1 mM ADPR ) clearly reflect spontaneous ADPR hydrolysis ( cf . , Figure 6—figure supplement 1 ) , because they remained insensitive to a ten fold variation in Chi4S or NUDT9H concentration ( Figure 6C , compare groups 4–5 and 6–7 ) , and were not larger than the [Pi] released in the absence of any ADPRase ( Figure 6C , group 3 ) . 10 . 7554/eLife . 17600 . 012Figure 6 . Enzymatic activity of NUDT9 , Chi4S , and NUDT9H measured with the Pi assay . ( A–B ) Pi ( in mM ) liberated in sample mixtures containing , as indicated , AP ( 5–6 U alkaline phosphatase ) , ADPR ( 1 mM ) , AMP ( 1 mM ) , R5P ( 1 mM ) , and either 5 or 50 nM protein ( NUDT9 , Chi4S , or NUDT9H ) , and incubated for 30 min at pH 6 . 5 , 7 . 1 , 8 . 5 , or 9 . 0 ( color coded ) at either 37°C ( A ) or room temperature ( RT ) ( B ) . Asterisks in A denote samples replotted with an expanded ordinate in Panel C . Bars represent mean ± SEM from 3 independent experiments . ( C ) Expanded display of samples denoted with asterisks in Panel A . ( D ) Estimated kcat values ( in s−1 ) for NUDT9 under each experimental condition ( pH and temperature ) , calculated ( see Materials and methods ) from [Pi] liberated in the samples containing 5 nM NUDT9 ( group 8 ) in Panels A and B . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 01210 . 7554/eLife . 17600 . 013Figure 6—figure supplement 1 . Spontaneous ADPR degradation at basic pH , monitored with Pi assay . Pi assay ( see Materials and methods ) for 2 mM ADPR in the absence of an ADPRase , incubated at various pH and temperatures ( as labeled ) for the indicated time ( in minutes ) . 'No AP' – Alkaline phosphatase was not added , and incubation time was 120 min . The two ordinates represent the concentration of liberated Pi ( in mM ) ( left ) , and the corresponding mol% of degraded ADPR ( right ) . Bars represent mean ± SEM from 3 independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 01310 . 7554/eLife . 17600 . 014Figure 6—figure supplement 2 . Size exclusion chromatograms of NUDT9 , HUDT9H , and Chi4S . Size exclusion chromatography elution profiles , detected as absorbance at 280 nm , of affinity-purified NUDT9 ( black ) , Chi4S ( blue ) , and NUDT9H ( red; note 10-fold expanded ordinate on the right ) . Peaks corresponding to monomers and dimers are labeledDOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 014 Importantly , lack of catalytic activity for Chi4S and NUDT9H was not due to microaggregation of these domains in a quasi-stable solution: size exclusion chromatograms for both proteins showed a main monomeric peak and a smaller peak corresponding to a dimer , just as for highly soluble NUDT9 ( Figure 6—figure supplement 2 , compare red and blue to black profile ) , while higher-stoichiometry aggregates constituted only a minor fraction in each case . For the above functional assays only the pooled fractions corresponding to the monomeric peaks were used . Enzymatic activity of purified Chi2 confirmed that it is a functional protein , similar to mitochondrial NUDT9 ( Figure 4A–B ) . To verify functionality also for enzymatically inactive Chi3 ( S ) and Chi4 ( S ) , we tested whether any of these constructs could substitute for the native NUDT9H domain in the context of full-length TRPM2 , to confer ADPR-induced gating upon the channel . For this purpose we used the non-inactivating ( Tóth and Csanády , 2012 ) T5L-TRPM2 pore mutant ( hereafter 'TRPM2' ) , and subcloned Chi2 , Chi3 and Chi4 into TRPM2 , replacing its native NUDT9H domain . The three full-length chimeric channels ( TRPM2-Chi2 , TRPM2-Chi3 and TRPM2-Chi4 ) were then expressed in Xenopus laevis oocytes , and tested for ADPR-dependent channel activation in inside-out patches . For all three constructs cytosolic superfusion with ADPR and Ca2+ triggered macroscopic currents ( Figure 7B–D ) resembling those of unmodified T5L-TRPM2 ( Figure 7A ) , indicating successful ligand binding to the chimeric domains: in 1 μM ADPR , the previously established half-saturating concentration for TRPM2 ( Tóth and Csanády , 2012 ) , similar fractional currents were activated for TRPM2 , TRPM2-Chi3 , and TRPM2-Chi4 ( Figure 7A , C–E ) , indicating similar apparent affinities for ADPR . Fractional activation of TRPM2-Chi2 by 1 μM ADPR was reduced ( Figure 7B , E ) , reflecting a modest , ~3 . 5-fold increase in the K1/2 for ADPR-induced current activation in TRPM2-Chi2 ( Figure 7H ) . The time constants of current decay upon ADPR removal were two–three-fold shorter for the chimeric constructs compared to TRPM2 ( Figure 7F ) , whereas the relaxation time constants upon Ca2+ removal were nearly identical ( Figure 7G ) . Overall , apart from moderate kinetic differences , all chimeric channel constructs were gated by ADPR similarly to TRPM2 , indicating that their chimeric ligand-binding domains are functional in the context of the full-length channel . 10 . 7554/eLife . 17600 . 015Figure 7 . ADPR-induced channel activity of full-length TRPM2 chimeras . ( A–D ) Macroscopic currents in inside-out patches excised from Xenopus laevis oocytes expressing ( A ) T5L-TRPM2 , ( B ) T5L-TRPM2-Chi2 , ( C ) T5L-TRPM2-Chi3 , and ( D ) T5L-TRPM2-Chi4 , elicited by superfusion with increasing concentrations of ADPR in the presence of saturating Ca2+ ( black bars ) . Membrane potential was −20 mV; current relaxation time courses following removal of either Ca2+ or ADPR were fitted by single exponentials ( colored lines ) with time constants indicated . ( E ) Fractional current activation of the constructs in A–D by 1 μM ADPR; mean steady currents in 1 μM ADPR were divided by those in 32 μM ADPR in the same patch ( mean ± SEM , n≥8 ) . ( F–G ) Time constants of current relaxation upon sudden removal of ADPR ( F ) or Ca2+ ( G ) for the constructs in A–D , obtained from single-exponential fits ( mean ± SEM , n≥5 ) . ( H ) Dose response curve of fractional current activation for T5L-TRPM2 ( gray ) and T5L-TRPM2-Chi2 ( light blue ) ; mean steady currents in various test [ADPR] were divided by those in 32 μM ADPR in the same patch . Symbols represent mean ± SEM ( n≥5 ) , solid lines are fits to the Hill equation with midpoints printed in the panel . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 015 Although the electrophysiological experiments confirm functionality for chimeric ligand binding domains in the context of a full-length TRPM2 channel expressed in frog oocytes , we sought to verify functionality also for Chi4S purified from E . coli , our best model of isolated NUDT9H . We found that the fluorescent ADPR analog 1 , N6-ethenoadenosine-5’-O-diphosphoribose ( ε-ADPR ) binds to the intact NUDT9H domain of full-length TRPM2 , because it suppressed ADPR-evoked TRPM2 currents in a dose-dependent manner: this effect was more pronounced at low , submaximal [ADPR] ( Figure 8A ) , suggesting a competitive inhibitory mechanism . Indeed , the dose response curve for ADPR-activation ( Figure 8B , blue symbols and fit line ) was right-shifted in the presence of 100 μM ε-ADPR ( Figure 8B , violet symbols ) , the fit ( Figure 8B , violet fit line ) suggesting a KI of 112 ± 24 μM for ε-ADPR binding to native NUDT9H . 10 . 7554/eLife . 17600 . 016Figure 8 . Full-length TRPM2 and the isolated Chi4S protein bind ε-ADPR with similar affinities . ( A ) Macroscopic inward T5L-TRPM2 current in inside-out patch excised from Xenopus laevis oocyte , elicited at -20 mV by repeated exposures to saturating Ca2+ ( black bars ) and 1 or 100 μM ADPR ( blue bars ) , in the absence or presence of 100 μM ε-ADPR ( red bars ) . ( B ) Dose-dependence of TRPM2 activation by ADPR in the absence ( blue symbols and fit line ) and presence ( violet symbols and fit line ) of 100 μM ε-ADPR ( mean ± SEM , n≥7 ) . Printed parameters KADPR , n , and Kε-ADPR reflect the apparent affinity and Hill slope for ADPR binding , and KI for ε-ADPR binding , respectively , obtained from the fits ( see Materials and methods ) . ( C ) Fluorescence intensity ( arbitrary units , Ex: 310 nm , Em: 420 nm ) of ε-ADPR at various total concentrations in the absence of protein ( black symbols ) , or in the presence of 100 μM of either BSA ( gray symbols ) or Chi4S ( red symbols ) . Symbols plot mean ± SD from four , two , and two independent titrations , respectively , for buffer ( black ) , BSA ( gray ) , and Chi4S ( red ) . Black line is a smooth fit of the black symbols by a three-parameter empirical mathematical function , and was used as a calibration curve to determine free [ε-ADPR] in the presence of Chi4S , assuming negligible fluorescence of the bound analog ( red projection lines ) . ( D ) Plot of upper estimate of free [ε-ADPR] as a function of total [ε-ADPR] in the presence of 100 μM Chi4S ( red symbols ) , fitted ( red line ) to a simple binding equation ( see Materials and methods ) to obtain an upper estimate of Kd ( see panel ) . Black line illustrates free [ε-ADPR] expected in the absence of binding . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 01610 . 7554/eLife . 17600 . 017Figure 8—figure supplement 1 . Lack of effect of five-fold concentration on Chi4S solubility . Size exclusion chromatography elution profiles , detected as absorbance at 280 nm . Chi4S was expressed and purified as described in Materials and methods , using Steptactin affinity chromatography followed by size exclusion chromatography ( solid blue profile ) . The pooled monomeric fraction was left overnight at 4°C , and then concentrated five-fold , to ~100 μM , using a 10 kDa molecular weight cutoff Vivaspin column ( Sartorius ) . This concentrated protein was then subjected to a second round of size exclusion chromatography ( dashed blue profile ) . DOI: http://dx . doi . org/10 . 7554/eLife . 17600 . 017 To test whether binding of ε-ADPR to a soluble protein might be revealed by a change in its fluorescence properties , we first calibrated the dose dependence of ε-ADPR fluorescence in free solution ( Figure 8C , black symbols and fit line; see Materials and methods ) . Applying 100 μM Chi4S caused a clear reduction in ε-ADPR fluorescence ( Figure 8C , red symbols ) , which reflected fluorescence quenching due to specific binding , because it was not observed upon addition of 100 μM bovine serum albumin ( BSA , gray symbols ) . Importantly , Chi4S showed no signs of aggregation even at the high protein concentration used in the assay ( Figure 8—figure supplement 1 ) excluding non-specific effects of aggregated protein on fluorescence . Assuming negligible fluorescence for bound ε-ADPR , a safe upper estimate of free [ε-ADPR] could be obtained from the calibration curve ( Figure 8C , red projection lines ) . A plot of free [ε-ADPR] as a function of total [ε-ADPR] ( Figure 8D , red symbols ) was reasonably fitted ( Figure 8D , red line ) by a simple binding equation , ( see Materials and methods ) yielding a Kd ( upper ) estimate of 111 ± 42 μM for ε-ADPR binding to Chi4S: in good agreement with the KI of the same analog for native NUDT9H ( Figure 8B ) .
The unique group of channel-enzymes ( chanzymes ) , transmembrane proteins that combine ion-channel function with a catalytic activity within a single polypeptide chain , includes the CFTR chloride ion channel , a member of the ATP Binding Cassette ( ABC ) protein family , and three members of the TRPM channel family , TRPM2 , 6 , and 7 . In the case of CFTR ATP hydrolysis by two cytosolic nucleotide binding domains ( NBDs ) ( Li et al . , 1996 ) appears to be an evolutionary vestige inherited from some ancestral ABC exporter ( Gadsby et al . , 2006 ) : whereas in transporters the energy of ATP hydrolysis is harnessed to drive a cycle of conformational changes that result in thermodynamically uphill substrate transport , in CFTR ATP-binding induced dimerization of the two NBDs is strictly coupled to opening , and dimer disruption following ATP hydrolysis to closure , of its transmembrane anion-selective pore ( Csanády et al . , 2010; Vergani et al . , 2005 ) . Such a nonequilibrium cyclic gating mechanism is unique among ion channels which mediate downhill electrodiffusion of ions , and confers specific properties on CFTR , including stimulation of channel open probability by catalytic-site mutations ( Vergani et al . , 2003 ) , or by drug-induced stabilization of energy barriers ( Csanády and Töröcsik , 2014 ) . The other well studied chanzyme is the TRPM7 cation channel , which contains a kinase domain fused to its cytosolic C-terminus ( Runnels et al . , 2001; Nadler et al . , 2001; Yamaguchi et al . , 2001 ) . In this protein the catalytic activity of the enzymatic domain does not appear to be linked to gating of the channel pore , because phosphotransferase and channel activities can be modulated independently of each other ( Matsushita et al . , 2005; Schmitz et al . , 2003 ) and the kinase domain is functional when expressed on its own ( Clark et al . , 2008 ) . Nevertheless , recent work uncovered unexpected important specific functions of TRPM7 enzymatic activity: the kinase domain is proteolytically cleaved and translocates to the cell nucleus where it regulates gene expression by phosphorylating histones in the promoters of TRPM7-dependent genes . Moreover , Zn2+ dependent binding of the kinase domain to histone remodeling complexes provides a functional link to the TRPM7 channel pore , a major Zn2+ influx pathway ( Krapivinsky et al . , 2014 ) . In the case of TRPM2 sequence similarity of its C-terminal NUDT9H domain to Nudix-family phosphohydrolases ( Bessman et al . , 1996 ) played an essential role in the discovery of the channel's activating ligand , ADP ribose ( Perraud et al . , 2001; Hara et al . , 2002; Sano et al . , 2001 ) . Early demonstration of ADPR hydrolase activity for the isolated ligand binding domain NUDT9H classified TRPM2 as a chanzyme ( Perraud et al . , 2001 , 2003 ) , and raised intriguing questions about the possible role of this activity: all the more as the reported slow turnover rate ( ~0 . 1 s-1 ) falls into the range of channel gating rates ( Tóth and Csanády , 2012 ) . Of note , while this slower rate of catalysis of NUDT9H , compared to NUDT9 , was explained by lack of a conserved glutamate in its Nudix box motif important for Mg2+ coordination , the actual catalytic base is highly variable in Nudix enzymes , and has not yet been identified for either of these two proteins ( Shen et al . , 2003 ) . However , when introduced into NUDT9H , catalytic site mutations that abolish , or greatly diminish , ADPRase activity of NUDT9 failed to affect the rates of pore gating , and TRPM2 channels were also readily gated by a non-hydrolyzable ADPR analog ( Tóth et al . , 2014 ) . These findings ruled out a direct link between the ADPRase cycle and gating of the TRPM2 channel pore itself , but did not eliminate the possibility of some , as yet unidentified , role for this catalytic function . The present work therefore addressed the putative enzymatic activity itself . To circumvent the problem of low solubility of NUDT9H we sought to establish a chimeric model which resembles the native protein sequence as closely as possible . The region responsible for the low solubility of NUDT9H could be confined to its ~60 C-terminal residues: replacing the entire span ( Chi3S ) , or even just the downstream half ( Chi4S ) of this C-terminal segment with homologous NUDT9 sequence ( Figure 3 ) was sufficient to confer solubility upon NUDT9H . The C-terminus of NUDT9 is surface exposed ( [Shen et al . , 2003]; PDB ID: 1Q33; 1QVJ ) , free to interact with other protein molecule ( s ) , and presumably the same is true for the C-termini of the chimeras and NUDT9H . Therefore , among other reasons , the cumulative charge of the C-terminus may play an important role in determining their aggregation propensity during expression in the bacterial cytoplasm ( at around neutral pH ) . The theoretical total charges of the ~60–70 C-terminal residues of our constructs ( ignoring affinity tags ) are −8 . 1 ( NUDT9 , Chi2 and Chi3 ) , +0 . 9 ( NUDT9H ) , −2 . 9 ( Chi4 ) at pH 7 . 0 . It is thus possible that the relative charge-neutrality of the C-terminus of NUDT9H contributes to its insolubility . Such low solubility , when expressed in isolation , for a cytoplasmic protein domain like NUDT9H – in striking contrast to its very soluble mitochondrial homologue – is somewhat unexpected . A likely explanation is that NUDT9H natively exists in the context of tetrameric TRPM2 , and must share an interface with other channel regions which transduces ligand binding into pore opening . Whether NUDT9H interacts with the large N-terminal domain of the channel , with the cytosolic loops connecting the transmembrane helices , and/or with other NUDT9H domains remains to be elucidated , but it is tempting to speculate that the C-terminal ~20% of NUDT9H sequence might be directly involved in such interactions . Consistent with such an interpretation , incremental replacement of NUDT9H segments with corresponding NUDT9 sequences ( TRPM2 ( -NUDT9H ) → TRPM2-Chi4 → TRPM2-Chi3 → TRPM2-Chi2 ) causes a parallel graded decrease in the stability of the open-channel state , as reflected by a speeding of channel closure upon ADPR removal ( Figure 7F; gray → red → dark blue → light blue bars ) . NUDT9 , the soluble chimeric constructs generated by sequence swapping , and even some NUDT9H could be purified and tested for enzymatic activity . These experiments revealed a distinct pattern directly correlated with the origin of the Nudix box in each protein: constructs possessing the Nudix box of NUDT9 ( NUDT9 and Chi2 ) were active ADPR hydrolases , whereas those containing the Nudix box of NUDT9H ( Chi3S , Chi4S and NUDT9H ) were inactive ( Figures 4 and 6 ) . Notably , our soluble chimeric model most similar to the native NUDT9H sequence , Chi4S , is ~90% identical to NUDT9H ( including the Nudix box region ) and clearly capable of ligand binding ( Figures 7D , 8C–D ) , but did not exhibit any sign of enzymatic activity under the various reaction conditions tested . Previous studies reported a weak enzymatic activity for the isolated NUDT9H domain of TRPM2 ( Perraud et al . , 2001 , 2003 ) . While we did also detect slow accumulation of AMP when Chi3S , Chi4S , or NUDT9H was incubated with ADPR under alkaline conditions , we fully attribute those results to inherent instability of ADPR at basic pH . Indeed , we observed gradual accumulation of AMP even in protein-free samples of ADPR incubated in alkaline buffers ( Figures 4F , 6C , Figure 4—figure supplement 2 , and Figure 6—figure supplement 1 ) and , assuming an exponential decay time course , estimated a time constant of ~100 hr for spontaneous ADPR hydrolysis at 25°C and pH 8 . 5 . Spontaneous degradation of ADPR at basic pH ( ~0 . 3% and ~2% within 20 min and 2 hr , respectively , at 25°C and pH 8 . 5; ~1% within 30 min at 37°C and pH 9 [Figure 6—figure supplement 1] ) did not significantly distort the results of the 20- to 30-min enzyme kinetics measurements for the active constructs ( Figures 5C and 6D ) , but fully accounted for the weak AMP signals for our inactive constructs observed after 2-hr incubations using TLC ( Figure 4C–E , lanes 3 and 6 ) , or already after 30-min incubations using the more sensitive Pi detection assay ( Figure 6A–C ) : this phenomenon fully explains the reported weak activity of NUDT9H at 37°C and pH 9 ( Perraud et al . , 2001 , 2003 ) . Of note , spontaneous degradation affects enzymatic ADPR hydrolysis assays only at basic , but not at neutral or slightly acidic pH at which ADPR is inherently stable ( Figure 4C–E , lanes 1 , 2 , 4 , 5; Figure 4—figure supplement 2 , and Figure 6—figure supplement 1 ) . The catalytic properties of our two enzymatically active recombinant proteins ( NUDT9 and Chi2 ) were largely similar . NUDT9 appeared slightly faster than Chi2 , which may be attributed either to slight structural mismatch between ‘foreign’ Cap and Core segments in Chi2 , or to errors in estimating their concentrations . Both enzymes exhibited a modest but clear pH-dependence , with a preference towards basic pH ( Figure 5C , and Figure 6D ) . Perhaps NUDT9 , which natively resides in mitochondria , might have adapted to this environment , and Chi2 , which contains ~60% of the NUDT9 sequence ( the entire Core subdomain ) , presumably shares most of its preferences . In contrast , the cytosolic NUDT9H domain of plasma-membrane resident TRPM2 faces the neutral pH of the cytosol . For this reason , care was taken to test the NUDT9H-like proteins ( Chi3S , Chi4S and NUDT9H ) in buffers at different pH values ( Figures 4 and 6 ) . When introduced into full-length TRPM2 in place of the native NUDT9H domain , chimeric constructs Chi2 , Chi3 and Chi4 supported ADPR-induced channel activity which resembled that of intact TRPM2 channels , except for a somewhat reduced apparent affinity of TRPM2-Chi2 ( Figure 7 ) . One possible reason for this small defect is that the other constructs contain catalytically inactive Nudix domains , whereas ADPR hydrolysis by Chi2 might reduce fractional saturation of this domain at low ( ~1 μM ) ADPR concentrations . Alternatively , the significantly larger portion of non-native ( i . e . NUDT9-like ) sequence in Chi2 ( the entire Core subdomain ) might compromise formation of the proper interface responsible for transducing ligand binding into channel opening . Nevertheless , all full-length chimeric TRPM2 constructs formed functional channels , demonstrating that NUDT9/NUDT9H chimeric domains are properly folded and competent to bind ADPR and gate the channel . Importantly , TRPM2-Chi4 is almost identical in sequence to the background T5L-TRPM2 , its channel behaviour is almost indistinguishable from that of T5L-TRPM2 , and we have clearly shown that its isolated ligand-binding domain ( Chi4S ) – although capable of binding an ADPR analog with a similar affinity as the intact NUDT9H domain of a full-length TRPM2 channel ( Figure 8 ) – is not catalytically active . Based on these results , we conclude that the NUDT9H domain of TRPM2 is not an ADPR hydrolase , and that TRPM2 is a simple ligand-gated channel , not a 'chanzyme' , despite the fact that its ligand binding domain is clearly a member of the family of Nudix enzymes . One potential advantage of its broken enzymatic activity might be increased ligand occupancy at low ADPR concentrations . The Chi4S construct developed here is a convenient soluble model of NUDT9H which might be useful for future structural and biochemical studies .
Except for Chimera 2 ( Chi2 ) , all recombinant proteins for bacterial expression were synthesized and incorporated into the pJ411 plasmid which bears kanamycin resistance ( DNA2 . 0 , Menlo Park , CA , USA ) . Chi2 was obtained by PCR-amplification of Chi2A cDNA using forward primer ( 5’-TTTAACTTTTAGGAGATAAACATATGCCGGATGCTGAG , NdeI site underlined ) and reverse primer ( 5’-AACGTCTGCTCGAGCAACGCATGGCAGTCAGCTTCAGA , XhoI site underlined ) , and subcloned into the pET21c vector . NUDT9 , NUDT9H , Chi1 and Chi2 had C-terminal hexahistidine tags , while Chi3S and Chi4S had C-terminal Twin-Strep tags . The N-termini of Chi1A , Chi2 , Chi2A , Chi3 , and Chi4 included an upstream stretch of 15 TRPM2 residues which contained a native BlpI-site ( Figure 3A , gray ) . The pJ411 plasmids harboring the individual constructs were transfected into inducible E . coli , strain BL21 ( DE3 ) . The noninactivating TRPM5-like pore mutant of TRPM2 ( T5L-TRPM2 ) was contained in the pGEMHE vector ( Tóth and Csanády , 2012 ) . The NUDT9H domain of TRPM2 was replaced by Chi2A , Chi3 or Chi4 using the native BlpI-site upstream of the domain and an XbaI-site downstream of the Stop-codon in each chimeric construct . Single-base mutations to generate Stop-codons immediately upstream of the hexahistidine tags of Chi3 and Chi4 were then introduced ( QuickChange II , Agilent Technologies , Santa Clara , CA , USA ) , yielding TRPM2-Chi3 and TRPM2-Chi4; TRPM2-Chi2 lacked a His-tag because it was derived from Chi2A . All constructs were sequence-verified . The TRPM2/pGEMHE plasmids were linearized with NheI ( New England Biolabs , Ipswich , MA , USA ) and transcribed in vitro using T7 polymerase; cRNA was stored at −80°C . The soluble proteins ( NUDT9 and Chi2 ) were expressed and purified as previously described for mitochondrial NUDT9 ( Tóth et al . , 2014 ) . In brief , bacterial cultures were grown at 37°C in Luria-Bertani medium supplemented with the appropriate antibiotic and induced with 1 mM isopropyl-β-D-1-thiogalactopyranoside ( IPTG ) upon reaching OD600 of ~0 . 7 . After 3 hr of incubation , cells were harvested and lysed with sonication in 20 mM Tris ( pH 8 . 5 with HCl ) / 200 mM NaCl . The cleared supernatant was incubated with Ni-Sepharose 6 Fast Flow resin ( GE Healthcare Hungary , Hungary ) for 3 hr under gentle rotation . The resin was then washed with lysis buffer supplemented with 20 mM imidazole , and the protein eluted with a buffer containing 20 mM Tris ( pH 8 . 5 with HCl ) , 100 mM NaCl , and 400 mM imidazole . Chi3S , Chi4S and NUDT9H were little soluble to insoluble when expressed at 37°C , the majority of the proteins ending up in inclusion bodies , but gave tractable ( ~1–5% ) soluble fractions when induced with 0 . 1 mM IPTG at 25°C overnight . NUDT9H was then purified as NUDT9 . Chi3S and Chi4S were purified using Strep-Tactin chromatography following the manufacturer’s instructions ( IBA GmbH , Göttingen , Germany ) . Following affinity chromatography , all proteins were subjected to a gel filtration purification step in 20 mM Tris ( pH 8 . 5 ) , 100 mM NaCl ( Superdex 200 10/300 GL , GE Healthcare Hungary ) . Affinity tags were not removed . Protein concentrations were determined spectrophotometrically using theoretical molar extinction coefficients ( εo ~ 55 , 000 M−1cm−1 at λ = 280 nm ) . Protein purity was visually checked with SDS PAGE . Protein identity was confirmed by the band position , the main peak position on the gel filtration profile , and , in the case of Chi4S , also by mass spectrometry ( Proteome Factory AG , Berlin , Germany ) . NUDT9 and Chi2 were flash-frozen in liquid nitrogen and kept at −20°C until used . Chi3S , Chi4S and NUDT9H were kept at 4°C , and immediately used for experiments . ADPRase activities of mitochondrial NUDT9 , NUDT9H , and the purified chimeric constructs ( Chi2 , Chi3S , Chi4S ) were quantitated by densitometric detection of the AMP product on TLC sheets . The 20-μl reaction mixtures contained 0 . 1 , 0 . 5 or 1 μM purified protein , 10 mM Na-ADPR ( Sigma-Aldrich , St . Louis , MO , USA ) , 16 mM MgCl2 , and 50 mM reaction buffer ( RB ) . The reaction buffers used were: 50 mM MES , pH 6 . 5 with NaOH ( RB1 ) ; 50 mM HEPES , pH 7 . 1 with NaOH ( RB2 ) ; 50 mM Tris , pH 8 . 5 with HCl ( RB3 ) ; RB1 + 100 mM NaCl ( RB4 ) ; RB2 + 100 mM NaCl ( RB5 ) , and RB3 + 100 mM NaCl ( RB6 ) . For the enzyme kinetics experiments on NUDT9 and Chi2 , aliquots from the reaction mixtures were collected at the indicated time points; 3 independent experiments were performed for both proteins and each condition , and data are presented as mean ± SEM . For other experiments , the total incubation time was as indicated in the figures legends . All incubations were at room temperature ( ~25°C ) . For TLC , 1-μl aliquots of reaction mixtures were placed on Polygram SIL G/UV254 plates ( Macherey-Nagel , Düren , Germany ) , dried and developed in 0 . 2 M NH4HCO3 in ethanol:water 7:3 ( vol/vol ) . Nucleotides were visualized under UV light . Densitometry of nucleotide intensities was done using ImageJ software . The ADPRase activities of the same proteins were additionally tested with a more sensitive assay based on colorimetric detection of Pi ( Pi assay ) liberated from both ADPRase products AMP and R5P by co-applied alkaline phosphatase ( AP ) ( Rafty et al . , 2002 ) . AP liberates Pi from AMP and R5P , but not from intact ADPR: thus , Pi is produced from hydrolyzed ADPR at a molar ratio of 1:2 . In brief , 150-μl reaction volumes containing 5 or 50 nM purified protein , 5–6 U bovine AP , 1 mM Na-ADPR , 16 mM MgCl2 , and 50 mM reaction buffer at various pH values ( MES 6 . 5 , HEPES 7 . 1 , Tris 8 . 5 , and Tris 9 . 0 ) were incubated for 30 min at either 37°C or room temperature . The reaction was stopped and Pi visualized by adding 850 μl coloring solution ( 6:1 vol/vol ratio mixture of 0 . 42% ammonium molybdate tetrahydrate in 1N H2SO4 and 10% L-ascorbic acid ) followed by incubation for 20 min at 45°C . Optical density was then measured at 820 nm ( NanoPhotometer P300 , Implen GmbH , Munich , Germany ) . Coloring solution was freshly made , and standard curve ( 0 . 050–2 mM KH2PO4 ) obtained , daily . Reactions with 1 mM AMP or R5P ( instead of ADPR ) served as positive controls . All reagents were from Sigma-Aldrich . Assays were performed in triplicates and data are displayed as mean ± SEM . Molecular turnover rates ( kcat ) for NUDT9 were calculated from the [Pi] released in samples containing 5 nM NUDT9 , in which [ADPR] remained >0 . 9 mM ( i . e . , saturating [Perraud et al . , 2003] ) throughout the 30-min incubation period , using the equation kcat = 0 . 5[Pi]released/5 nM/1800 s . The ligand binding ability of purified Chi4S protein was assessed using fluorescence quenching of the ADPR analogue 1 , N6-ethenoadenosine-5’-O-diphosphoribose ( ε-ADPR - BIOLOG Life Science Institute , Bremen , Germany ) . A 350-μl volume of 100 μM Chi4S protein in 20 mM Tris ( pH 8 . 5 ) /100 mM NaCl buffer , supplemented with 16 mM MgCl2 , was titrated by increasing concentrations of ε-ADPR . Fluorescence emission scans ( 350–700 nm ) were recorded at a fixed excitation wavelength of 310 nm ( Hitachi F-7000 , Hitachi High Technologies , Maidenhead , UK ) . Fluorescence emission for ε-ADPR peaks at ~410 nm . Protein-free buffer or 100 μM bovine serum albumin ( BSA; instead of Chi4S ) were used as controls . Data are shown as mean ± S . D . of two independent experiments . Dose dependence of fluorescence ( Em = 420 nm ) of free ε-ADPR ( in buffer ) was fitted by an empirical non-linear function to produce a smooth calibration curve , used to obtain upper estimates of free [ε-ADPR] in the presence of 100 μM Chi4S protein and various total [ε-ADPR] , assuming negligible fluorescence for the bound , relative to the free , form of the analog . The plot of free vs . total [ε-ADPR] was fitted by the simple binding equation[ε-ADPR]free=0 . 5 ( [ε-ADPR]total−[P]total−Kd+ ( [ε-ADPR]total−[P]total−Kd ) 2+4Kd[ε-ADPR]total ) , where [P]total is the total concentration of Chi4S protein ( 100 μM ) , and Kd is a free parameter . Given that the values of free [ε-ADPR] are upper estimates , this approach provides an upper estimate of the Kd . Because for the binding assay the Chi4S preparation had to be concentrated by ~five-fold to reach ~100 μM ( 3 . 6 mg/ml ) , the effect of such a concentration step on Chi4S solubility was verified by subjecting five-fold concentrated Chi4S protein to a second round of size exclusion chromatography . The elution profile of this second run ( Figure 8—figure supplement 1 , dashed blue profile ) resembled that of the first run , showing a main monomeric peak with a smaller peak corresponding to a dimer . Importantly , larger aggregates did not reappear . This confirms that the signal observed in the fluorescent binding assay is not due to non-specific effects of aggregated protein . Oocytes isolated from anaesthetized Xenopus laevis were digested with collagenase , injected with 10–70 ng cRNA , and stored at 18°C . Recordings were done 2–3 days after injection . The tip of the patch pipette was filled to ~1 cm height with 140 mM Na-gluconate , 2 mM Mg-gluconate2 , 10 mM HEPES ( pH 7 . 4 with NaOH ) ; free [Ca2+] ~4 μM . The pipette electrode was placed into a 140-mM NaCl-based solution carefully layered on top . Bath solution contained 140 mM Na-gluconate , 2 mM Mg-gluconate2 , 10 mM HEPES ( pH 7 . 1 with NaOH ) , and either 1 mM EGTA ( to obtain 'zero' ( ~8 nM ) Ca2+ ) or 1 mM Ca-gluconate2 ( to obtain 125 μM free Ca2+ ) ( Csanády and Törocsik , 2009 ) . Na-ADPR was used at final concentrations of 0 . 32 , 1 , 3 . 2 , 10 , 32 , 100 , or 1000 μM , and the Na+ salt of ε-ADPR at 100 μM . Macroscopic TRPM2 currents were recorded at 25°C at a membrane potential of −20 mV . Currents were amplified , filtered at 2 kHz ( Axopatch 200B ) , digitized at 10 kHz ( Digidata 1322A ) , and stored to disk ( Pclamp9 , Molecular Devices , Sunnyvale , CA , USA ) . Dose dependence of ADPR-induced current activation , expressed as the ratio of mean macroscopic currents observed in the presence of various test [ADPR] ( I ) and 32 ( or 100 ) μM ADPR ( Imax ) in the same patch , was fitted to the Hill equation ( I/Imax = [ADPR]n/ ( Kn+[ADPR]n ) . Dose dependence of ADPR-activation in the presence of 100 μM ε-ADPR was expressed as the ratio of the currents observed in the presence of various test [ADPR] + 100 μM ε-ADPR ( I ) and 100 μM ADPR alone ( Imax ) in the same patch , and was fitted to the Hill equation substituting K' = K ( 1+[ε-ADPR]/KI ) instead of K ( K and n fixed to their control values ) . Macroscopic current decay time courses following removal of ADPR or Ca2+ were fitted by single exponentials using the method of least squares . Data are displayed as mean ± SEM from n independent experiments , as specified in each figure legend . All data were included in the analysis . Enzyme activities were assayed in at least three independent experiments . Gating parameters for full length TRPM2 and chimeric channels were estimated from current recordings obtained from at least five patches in each case . These numbers of technical replications allowed us to limit SEM to ≤20% of the mean for each measured parameter .
Transient Receptor Potential Melastatin 2 ( TRPM2 ) is a Ca2+-permeable cation channel essential for immunocyte activation , insulin secretion , and postischemic cell death . TRPM2 is activated by ADP ribose ( ADPR ) binding to its C-terminal cytosolic NUDT9-homology ( NUDT9H ) domain , homologous to the soluble mitochondrial ADPR pyrophosphatase ( ADPRase ) NUDT9 . Reported ADPR hydrolysis classified TRPM2 as a channel-enzyme , but insolubility of isolated NUDT9H hampered further investigations . Here we developed a soluble NUDT9H model using chimeric proteins built from complementary polypeptide fragments of NUDT9H and NUDT9 . When expressed in E . coli , chimeras containing up to ~90% NUDT9H sequence remained soluble and were affinity-purified . In ADPRase assays the conserved Nudix-box sequence of NUDT9 proved essential for activity ( kcat~4-9s-1 ) , that of NUDT9H did not support catalysis . Replacing NUDT9H in full-length TRPM2 with soluble chimeras retained ADPR-dependent channel gating ( K1/2~1-5 μM ) , confirming functionality of chimeric domains . Thus , TRPM2 is not a 'chanzyme' . Chimeras provide convenient soluble NUDT9H models for structural/biochemical studies .
Ion channels are proteins that allow specific charged particles to move across the membranes of cells – for example to travel in or out of a cell , or between different parts of the same cell . Almost all ion channels are gated , meaning that they can open and close in response to different signals . For instance , so-called ligand gated channels open in response to binding of some small molecule , known as the "ligand" . A small number of channel proteins are also enzymes , meaning that they are able to catalyze chemical reactions , and these channel-enzymes are often referred to as “chanzymes” . TRPM2 is an ion channel found in humans that opens when a small molecule called ADPR binds to a portion of the channel inside the cell . This channel is also thought to be a chanzyme because the part that binds to ADPR is similar to an enzyme called NUDT9 . The NUDT9 enzyme converts ADPR into two other chemicals . When studied in biochemical assays , the enzyme-like part of TRPM2 – which contains a segment called a “Nudix box” – appeared to act in the same way , although this activity was not linked to the opening and closing of the TRPM2 channel . Iordanov et al . set out to re-examine whether TRPM2 is actually an enzyme by comparing the activity of NUDT9 to the enzyme-like part of TRPM2 . To test an enzyme’s activity , it typically needs to be dissolved in water . However , the enzyme-like part of TRPM2 does not dissolve , and so it could not be tested directly . Instead , Iordanov et al . identified which parts of TRPM2 make it insoluble and replaced them with the equivalent parts from NUDT9 to create several new proteins . For all the proteins tested , only those with the Nudix box from NUDT9 were active enzymes , while those with the Nudix box from TRPM2 were not . Iordanov et al . conclude that TRPM2 is a ligand gated channel and not a chanzyme , and that the experimental conditions used in previous biochemical assays , and not TRPM2 activity , led to the breakdown of ADPR . Finally , the TRPM2 channel is involved in cell damage following heart attacks or stroke and may contribute to Alzheimer’s disease , Parkinson’s disease and bipolar disorder as well . As such , knowing how TRMP2 behaves could guide efforts to develop new drugs for these illnesses .