NtCDPK1 homodimerization is decreased by autophosphorylation of either Ser-6 or Thr-21.

<p>Autophosphorylated and unphosphorylated GST-NtCDPK1 were immobilized on glutathione beads. After washing, GST-NtCDPK1-immobilized beads were incubated with His-NtCDPK1. GST-NtCDPK1-bound proteins were subjected to SDS-PAGE, followed by immunoblotting with anti-NtCDPK1 antibody for the detection of His-NtCDPK1. GST-NtCDPK1 proteins were visualized by CBB staining. This experiment was repeated three times with similar results.</p

Ser-6 and Thr-21 are autophosphorylated by different mechanisms.

<p>Phosphorylation of GST-NtCDPK1 proteins by His-NtCDPK1 was by reacting GST-NtCDPK1 WT, GST-NtCDPK1 D219N, GST-NtCDPK1 S6A/D219N, GST-NtCDPK1 T21A/D219N, and GST-NtCDPK1 S6A/T21A/D219N with His-NtCDPK1 WT in autophosphorylation buffer. The autophosphorylation state of GST-NtCDPK1 proteins was examined using Phos-tag SDS-PAGE. GST-NtCDPK1 proteins were detected by immunoblotting with anti-GST antibody. His-NtCDPK1 WT was visualized by CBB staining. Letters represent phosphorylated GST-NtCDPK1 WT (a), unphosphorylated GST-NtCDPK1 WT, GST-NtCDPK1 S6A/D219N, and GST-NtCDPK1 S6A/T21A/D219N (b), and phosphorylated GST-NtCDPK1 D219N and GST-NtCDPK1 T21A/D219N (c). This experiment was repeated three times with similar results.</p

Ser-6 of NtCDPK1 is autophosphorylated faster than Thr-21.

<p>GST-NtCDPK1 WT, GST-NtCDPK1 S6A, and GST-NtCDPK1 T21A were autophosphorylated for the indicated time periods. The autophosphorylation state of GST-NtCDPK1 proteins was examined using Phos-tag SDS-PAGE. GST-NtCDPK1 proteins were visualized by Coomassie Brilliant Blue (CBB) staining. This experiment was repeated three times with similar results.</p

Autophosphorylation of Ser-6 and Thr-21 leads to rapid and slow inhibition of phosphorylation of RSG, respectively.

<p>(A) MBP-RSG was phosphorylated for the indicated time periods. Phosphorylation of Ser-114 of RSG was detected by immunoblotting using anti-pS114 antibody, which specifically recognizes phosphorylated Ser-114 of RSG. MBP-RSG was visualized by CBB staining. GST-NtCDPK1 proteins were detected by immunoblotting with anti-GST antibody. This experiment was repeated three times with similar results. (B) Statistical analysis of the data shown in (A). The quantified band intensity of phosphorylated RSG by GST-NtCDPK1 S6A/T21A at 5 min (left panel) and 60 min (right panel) was set to 1, respectively. The bar graph represents means and ± SE (<i>n</i> = 3). Significant differences in the phosphorylation of MBP-RSG by GST-NtCDPK1 proteins are determined by one-way ANOVA with Tukey’s honestly significant difference test at each time point. Different letters above the bars indicate significant differences between the relative intensities at each time point (<i>P</i> < 0.05). (C) Line graph represents the band intensities of phosphorylated RSG obtained from (A) and (B).</p

Identification of the Binding Position of Amilorides in the Quinone Binding Pocket of Mitochondrial Complex I

We previously demonstrated that amilorides bind to the quinone binding pocket of bovine mitochondrial complex I, not to the hitherto suspected Na⁺/H⁺ antiporter-like subunits (ND2, ND4, and ND5) [Murai, M., et al. (2015) <i>Biochemistry</i> <i>54</i>, 2739–2746]. To characterize the binding position of amilorides within the pocket in more detail, we conducted specific chemical labeling [alkynylation (−CCH)] of complex I via ligand-directed tosyl (LDT) chemistry using a newly synthesized amide-type amiloride AAT as a LDT chemistry reagent. The inhibitory potency of AAT, in terms of its IC₅₀ value, was markedly higher (∼1000-fold) than that of prototypical guanidine-type amilorides such as commercially available EIPA and benzamil. Detailed proteomic analyses in combination with click chemistry revealed that the chemical labeling occurred at Asp160 of the 49 kDa subunit (49 kDa Asp160). This labeling was significantly suppressed in the presence of an excess amount of other amilorides or ordinary inhibitors such as quinazoline and acetogenin. Taking into consideration the fact that 49 kDa Asp160 was also specifically labeled by LDT chemistry reagents derived from acetogenin [Masuya, T., et al. (2014) <i>Biochemistry</i> <i>53</i>, 2307–2317, 7816–7823], we found this aspartic acid to elicit very strong nucleophilicity in the local protein environment. The structural features of the quinone binding pocket in bovine complex I are discussed on the basis of this finding

Amilorides Bind to the Quinone Binding Pocket of Bovine Mitochondrial Complex I

Amilorides, well-known inhibitors of Na⁺/H⁺ antiporters, were previously shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I) but were markedly less active for complex I. Because membrane subunits ND2, ND4, and ND5 of bovine complex I are homologous to Na⁺/H⁺ antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is currently no direct experimental evidence that supports this notion. To identify the binding site of amilorides in bovine complex I, we synthesized two photoreactive amilorides (PRA1 and PRA2), which have a photoreactive azido (-N₃) group and terminal alkyne (-CCH) group at the opposite ends of the molecules, respectively, and conducted photoaffinity labeling with bovine heart submitochondrial particles. The terminal alkyne group allows various molecular tags to covalently attach to it via Cu⁺-catalyzed click chemistry, thereby allowing purification and/or detection of the labeled peptides. Proteomic analyses revealed that PRA1 and PRA2 label none of the antiporter-like subunits; they specifically label the accessory subunit B14.5a and core subunit 49 kDa (N-terminal region of Thr25–Glu115), respectively. Suppressive effects of ordinary inhibitors (bullatacin, fenpyroximate, and quinazoline), which bind to the putative quinone binding pocket, on labeling were fairly different between the B14.5a and 49 kDa subunits probably because the binding positions of the three inhibitors differ within the pocket. The results of this study clearly demonstrate that amilorides inhibit complex I activity by occupying the quinone binding pocket rather than directly blocking translocation of protons through the antiporter-like subunits (ND2, ND4, and ND5). The accessory subunit B14.5a may be located adjacent to the N-terminal region of the 49 kDa subunits. The structural features of the quinone binding pocket in bovine complex I were discussed on the basis of these results

Production of new amilorides as potent inhibitors of mitochondrial respiratory complex I

<div><p>Amilorides, well-known inhibitors of Na⁺/H⁺ antiporters, have also shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I). Since the membrane subunits ND2, ND4, and ND5 of bovine mitochondrial complex I are homologous to Na⁺/H⁺ antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is no direct experimental evidence in support of this notion. Photoaffinity labeling is a powerful technique to identify the binding site of amilorides in bovine complex I. Commercially available amilorides such as 5-(<i>N</i>-ethyl-<i>N</i>-isopropyl)amiloride are not suitable as design templates to synthesize photoreactive amilorides because of their low binding affinities to bovine complex I. Thereby, we attempted to modify the structures of commercially available amilorides in order to obtain more potent derivatives. We successfully produced two photoreactive amilorides (PRA1 and PRA2) with a photolabile azido group at opposite ends of the molecule.</p></div

Production of new amilorides as potent inhibitors of mitochondrial respiratory complex I

<div><p>Amilorides, well-known inhibitors of Na⁺/H⁺ antiporters, have also shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I). Since the membrane subunits ND2, ND4, and ND5 of bovine mitochondrial complex I are homologous to Na⁺/H⁺ antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is no direct experimental evidence in support of this notion. Photoaffinity labeling is a powerful technique to identify the binding site of amilorides in bovine complex I. Commercially available amilorides such as 5-(<i>N</i>-ethyl-<i>N</i>-isopropyl)amiloride are not suitable as design templates to synthesize photoreactive amilorides because of their low binding affinities to bovine complex I. Thereby, we attempted to modify the structures of commercially available amilorides in order to obtain more potent derivatives. We successfully produced two photoreactive amilorides (PRA1 and PRA2) with a photolabile azido group at opposite ends of the molecule.</p></div

Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction

We previously showed that a bulky ring-strained cycloalkyne possessing a rhodamine fluorophore directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine heart submitochondrial particles [Masuya, T., et al. (2014) <i>Biochemistry</i> <i>53</i>, 7816–7823]. This two-step conjugation may be a promising technique for specific chemical modifications of the quinone-access channel in complex I by various molecular probes, which would lead to new methodologies for studying the enzyme. However, because the reactivities of ring-strained cycloalkynes are generally high, they also react with other nucleophilic amino acids in mitochondrial proteins, resulting in significant undesired side reactions. To minimize side reactions and achieve precise pinpoint chemical modification of 49 kDa Asp160, we investigated an optimal pair of chemical tags for the two-step conjugation reaction. We found that instead of strain-promoted click chemistry, Diels–Alder cycloaddition of a pair of cyclopropene incorporated into 49 kDa Asp160 (via ligand-directed tosyl chemistry) and externally added tetrazine is more efficient for the pinpoint modification. An excess of quinone-site inhibitors did not interfere with Diels–Alder cycloaddition between the cyclopropene and tetrazine. These results along with the previous findings (cited above) strongly suggest that in contrast to the predicted quinone-access channel modeled by X-ray crystallographic and single-particle cryo-electron microscopic studies, the channel is open or undergoes large structural rearrangements to allow bulky ligands into the proximity of 49 kDa Asp160