Backbone resonance assignments of dopamine N-acetyltransferase in free and cofactor- bound states

doi:10.21203/rs.3.rs-5453819/v1

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Backbone resonance assignments of dopamine N-acetyltransferase in free and cofactor- bound states

https://doi.org/10.21203/rs.3.rs-5453819/v1

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published 12 Feb, 2025

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Dopamine N-acetyltransferase (Dat), belonging to the GCN5-related N-acetyltransferase (GNAT) superfamily, is an arylalkylamine N-acetyltransferase (AANAT) that involved in insects neurotransmitter inactivation and the development of insect cuticle sclerotization. By using the cofactor acetyl coenzyme A (Ac-CoA) as an acetyl group donor, Dat produces acetyl-dopamine through the catalysis of dopamine. Although AANATs share similar structural features with the GNAT family, they have low sequence identities among insect AANATs (~ 40%) and between insect AANATs and vertebrate AANATs (~ 12%). A common noticed feature in GNATs is the Ac-CoA-binding induced conformational change, and is important for further selection and catalysis of its substrate. In AANATs, the conformational changes help the sequential binding mechanism. Here, we finished the ¹H, ¹³C and ¹⁵N backbone resonance assignments of the 24 kDa Dat from Drosophila melanogaster in the free and Ac-CoA-bound states, and the chemical shift differences revealed a significant conformational change in the α1 region of Dat. These assignments provide a foundation for further investigations of the catalysis and structural regulation of Dat in solution.

Arylalkylamine N-acetyltransferases (AANATs) that have been identified in vertebrates (Ferry et al., 2004; Namboodiri et al., 1987) and invertebrates (Brodbeck et al., 1998; Mehere et al., 2011) use acyl-CoA (usually Ac-CoA) as an acyl-group donor for the N-acetylation of monoamines and have important functions in bodies. In vertebrates, the major role of AANATs was found to be in the melatonin synthesis pathway, in which AANATs control the day/night rhythm in melatonin production. Vertebrate AANATs are expressed in the pineal gland, retina, and parietal eyes, which are photosensitive, and transfer acetyl group from Ac-CoA to serotonin to produce N-acetyl-serotonin. N-Acetyl-serotonin can then be further converted to melatonin by hydroxyindole-O-methyltransferase (HIOMT). In daytime, the activity of AANAT is low, resulting in low levels of N-acetyl-serotonin and melatonin. However, in nighttime, AANAT shows increased activity and elevated N-acetyl-serotonin and melatonin production. Thus, the vertebrate AANAT is called “the Timezyme” (Klein, 2007). The AANAT gene was also found to be related to numerous genetic diseases, such as delayed sleep phase syndrome (DSPS), in patients who have higher percentage of Ala129Thr mutation than normal individuals (Hohjoh et al., 2003). Given the close association between melatonin production and sleep regulation as well as emotional processes (Yang et al., 2021), recent studies have increasingly identified AANAT as a potential drug target (Hagemeister et al., 2023).

In contrast, insects have several AANATs that have been suggested to participate in multiple functions (Han et al., 2012). In addition, insect AANATs are expressed widely in organisms, such as in the brain, ventral nerve cord, optic lobes, midgut, and eggs, and can catalyze monoamines, including dopamine, tyramine, serotonin, and octopamine (Brodbeck et al., 1998; Mehere et al., 2011; Tsugehara et al., 2007). Recently, a study found that the AANAT in Aedes aegypti can also catalyze the secondary amine, epinephrine (Zhang et al., 2022). To date, three major physiological functions of insect AANATs have been identified. First, insect AANATs have been suggested to play a key role in neurotransmitter inactivation by the catabolism of catecholamines, because the major enzyme in vertebrates that catalyzes catecholamines, monoamine oxidase (MAO), is expressed at low levels in insects (Dewhurst et al., 1972; Hiragaki et al., 2015; Sloley, 2004). Second, all insects have cuticles, and the production of N-acetyl-dopamine by AANATs could be a material of sclerotization (Andersen, 2010). Knockdown of AANAT would affect exoskeleton development and the deposition of melanin (Long et al., 2015; Noh et al., 2016). Recent study in Aedes aegypti has highlighted the role of AANAT7 in reproduction and adult limb pigmentation (Tang et al., 2024). Finally, some insect AANATs, such as AANATL2 in Drosophila melanogaster (Dempsey, Jeffries, Anderson, et al., 2014) and AANAT in Bombyx mori (Anderson et al., 2018), were identified to have a potential role in the biosynthesis of fatty acid amides, to which AANATs can transfer acyl groups and produce N-fatty acylamides. A special case showed that insect AANATs would participate in the circadian system in Antheraea pernyi (Mohamed et al., 2014) by the evidence that turning off the transcription of aanat caused photoperiodism dysfunction. Owing to the functional diversity and low sequence identity among insect AANATs (~ 40%), it is meaningful to distinguish their different roles as well as the substrate specificity of AANATs. Therefore, insect AANATs were recently considered potential targets for insecticide investigations to produce compounds that would specifically affect the activities of insect rather than vertebrates (O'Flynn et al., 2018; O'Flynn et al., 2020; Tsugehara et al., 2013; Zhang et al., 2023).

Although there are low sequence identities and different substrate specificities among insect AANATs and between insect and vertebrate AANATs, they belong to the GCN5-related N-acetyltransferase (GNAT) superfamily which have conserved structural features, motifs A-D (Dyda et al., 2000; Ud-Din et al., 2016), and maybe have similar binding mode or protein conformational regulation. Many researchers have found that the binding of cofactors or substrates could change the conformations of GNAT proteins. hNaa50p (hNAT5/hSAN), which transfers the acetyl group onto the N-terminal methionine residue, a protein conformational change upon Ac-CoA binding was observed, and the flexibility of the central β4 might be an essential requirement for the peptide to bind (Evjenth et al., 2012). In Saccharomyces cerevisiae GNA1, which involved in UDP-GlcNAc biosynthesis, Ac-CoA binding induced slight structural rearrangements at the edges of the cleft, loops (α3β5 and β5α4) and N-cap of α4, and caused a slightly narrower cleft (Peneff et al., 2001). In AANATs, much evidence also suggests that there is a conformational change during cofactor binding to fit the substrate and supports the ordered sequential binding mechanism. Crystal structures of SNAT (Oris aries; PDB codes: 1CJW and 1B6B) showed the loop-to-helix transition of α1 in the bisubstrate analog complex compared to the apo form (Hickman et al., 1999). Crystal structures of Dat (Dopamine N-acetyltransferase in D. melanogaster; PDB codes: 3V8I, 3TE4, and 5GI9) in the apo, binary, and ternary forms revealed that the most significant conformational changes occurred from the free state to the cofactor-bound state (Wu et al., 2020). In AANAT3 from Bombyx mori, the binding of Ac-CoA could regulate the conformational properties of both the cofactor and substrate binding sites (Aboalroub, Bachman, et al., 2017); however, a large region of the protein showed conformational heterogeneity in both the apo- and ligand-bound states, and the role of the different conformers and their dynamic properties in the catalytic cycle remain unknown (Aboalroub, Zhang, et al., 2017). Solution NMR studies of AANATs would help to investigate the substrate selection and protein dynamic properties of enzyme catalysis. Here, we present backbone resonance assignment of Dat in the free and Ac-CoA-bound states, providing a basis for the dynamic study of the regulation of these enzymes in substrate binding, release, and selection.

Expression and purification of Dat

A truncated Dat, Dat_{21 − 230}, was used to remove disorder region in N-terminus and C-terminus (Cheng et al., 2012). This truncated sequence is consistent with the published crystal structures that exhibited a similar function and secondary structure to that of full-length Dat (Cheng et al., 2012). Dat_{21 − 230} was cloned into a pET28a vector with an N-terminal His-tag, a TEV cleavage site, and kanamycin resistance and was expressed in Escherichia coli BL21(DE3) cells. Cells were grown in M9 medium containing ¹⁵N-NH₄Cl and ¹³C-glucose at 37°C until the OD₆₀₀ reached 0.7, and then protein expression was induced by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25°C for 16 hours. Cells were harvested by centrifugation at 6,000×g at 4°C for 30 min, resuspended in buffer A (50 mM Tris − HCl, 100 mM NaCl, 14 mM β-mercaptoethanol, pH 8.0) and lysed by sonication. Cell debris were removed by centrifugation at 16,000×g and 4°C for 25 min. The 0.22 µm-filtered lysate was loaded on a Ni²⁺ column (GE Healthcare), washed, and eluted with buffer A containing 20 mM and 500 mM imidazole, respectively. The eluent containing Dat_{21 − 230} was dialyzed against buffer A and then was treated with TEV protease to remove the N-terminal His-tag. After cleavage, Dat was further purified with a 1 ml HiTrap Q XL column (GE Healthcare) with a gradient concentration of NaCl from 0.1 to 1 M in buffer A followed purification on a Superdex 75 column (GE Healthcare) with buffer B (50 mM Tris − HCl, 100 mM NaCl, 1 mM DTT, and 1 mM EDTA, pH 7.0). Purified Dat was concentrated to 24 mg/ml, flash-frozen in liquid nitrogen, lyophilized, and stored at -80°C.

NMR spectroscopy

The lyophilized ¹⁵N, ¹³C-labeled protein was dissolved in H₂O/D₂O, and the final buffer conditions were 50 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, and 1 mM EDTA in 90% H₂O/10% D₂O containing 0.2 mM DSS (2,2-dimethyl-2-silapentane-5-sulfonate). All NMR experiments were performed with 1 mM Dat at 20°C for apo-Dat and 25°C for the Dat/Ac-CoA binary complex (in a ratio of 1:4) and data were collected on an Avance III HD 850-MHz NMR spectrometer (Bruker, Germany) equipped with a ¹H/¹³C/¹⁵N cryoprobe. Non-uniform sampled (NUS) 3D-heteronuclear NMR experiments, HNCO, HNCACO, HNCA, HNCOCA, HNCB, and HNCACB were acquired for the sequential assignments of ¹H, ¹⁵N, and ¹³C backbone chemical shifts. Assignments were confirmed and improved by selective unlabeling of different types of amino acids. Amino acid selective unlabeling can provide the exact amino acid types; hence, it can accelerate to deal with the connections of each residue and rule out the alternative possibility of a connection. Spectra were processed by NMRpipe (Delaglio et al., 1995) and analyzed with NMRFAM-SPARKY (Lee et al., 2015). Chemical shift differences, Δδ, were calculated according to the equation below, where α was 0.14 for most residues but 0.2 for glycine (Williamson, 2013):

$$\:{\Delta\:}{\delta\:}=\sqrt{\frac{1}{2}\:[{{\delta\:}}_{H}^{2}+\left(\alpha\:\bullet\:{{\delta\:}}_{N}^{2}\right)]}\:$$

Assignments and data deposition

The ¹⁵N-¹H HSQC spectra of apo-Dat and Dat/Ac-CoA are shown in Fig. 1. Out of the 197 non-proline residues, the amide signals of 184 residues (93.4%) and 192 residues (97.5%) were assigned in apo-Dat and in Dat/Ac-CoA, respectively, with complete or partial C’, Cα, and Cβ resonances. We also confirmed the assignments by amino acid selective unlabeling. The missing assignments in apo-Dat were N50, K59, Y75, I85, M93, G154, G156, I157, H184, F195, Q209, G210, and E211, in which G154, G156, and I157 are in the P-loop sequence (R153-A158). The P-loop, which is the sequence Gln/Arg-x-x-Gly-x-Gly/Ala, is a conserved signature in GNAT proteins that is responsible for Ac-CoA binding (Ud-Din et al., 2016). While, unassigned I85 is in the loop before β3, which has two backbone hydrogen bonds with A77 (on β2) and D149 (on the loop before the P-loop sequence). Another set of missing assignments of the consecutive residues Q209, G210, and E211 was located at the loop connecting α8 and β7. Unassigned N50 is at the α1 terminus, which provides two hydrogen bonds to the hydroxyl groups of dopamine and was suggested to be responsible for the binding of dopamine. In contrast, most of these unassigned residues could be assigned in the Dat/Ac-CoA binary complex, and the missing resonances in Dat/Ac-CoA were S182, H184, E191, K192, and K215. Most of these residues are located in α7 (184–192), except K215, which is located at the loop between α8 and α9, and S182, which is located at the loop adjacent α7 and is one of the catalytic residues (Cheng et al., 2012). K192 can form two salt bridges to the 3’-adenosine phosphate and pyrophosphate groups of Ac-CoA. The missing resonances of the P-loop in the free state that are shown in the Ac-CoA-bound state revealed that the binding of Ac-CoA reduced the fluctuation of the P-loop region. The loss of the resonance of S182 in the Ac-CoA-bound state implied a conformational exchange on the millisecond timescale, which might be related to catalysis, as the turnover rate of Dat is 17.6 s^− 1 for dopamine (Dempsey, Jeffries, Bond, et al., 2014).

Chemical shift predicted secondary structure using TALOS-N (Shen & Bax, 2013) showed that apo-Dat was similar to Dat/Ac-CoA, and both were consistent with the secondary structures found in the crystal structures (PDB code: 3V8I and 3TE4 for apo-Dat and Dat/Ac-CoA, respectively), except for a new β-strand at the C-terminus of Dat/Ac-CoA (Fig. 2). The short α5 (near the P-loop) and α9 defined in the crystal structures were predicted as loops in solution states. The predicted order parameters (S² values) calculated from the RCI values showed that all the flexible regions were consistent with the loop regions of the crystal structures (Fig. 2). A significant flexible loop (N79-I85) connecting β2 and β3 was observed and presented conformational heterogeneity in both the apo-Dat and the Dat/Ac-CoA complex. In the Dat/Ac-CoA complex, E83 and I84 showed two conformers with a relative ratio of intensity of ~ 80:20. Another resonance near the P-loop sequence, N151, presented the same proportion of intensities of two signals. Among these three resonances, E83 showed much differences in the two conformations because relatively large differences in ¹H-¹⁵N chemical shift correlations were found (~ 0.2 ppm of ¹H resonance) (Fig. 1). Similar conformational heterogeneity was also found in apo-Dat, in which a minor conformer of I84 was found (relative ratio of ~ 85:15). Additionally, two hydrogen bonds formed within the side chains of the residues E83-N79-Y152 in the crystal structures, which connect the flexible loop (N79-I85) and the loop adjacent to the P-loop sequence. It seems that conformational heterogeneity simultaneously exists in the two loops but is independent of the binding of Ac-CoA because they exist in both the free and bound states of Dat.

The similarity in the predicted secondary structures for the apo-Dat and Dat/Ac-CoA binary complex revealed no major structural changes upon binding. However, the chemical shift differences indicated significant changes in α1, β4-P-loop-α6 and α7 (Fig. 3), suggesting conformational changes between these two forms. According to the crystal structure, the binding site of Ac-CoA was major at β4, P-loop, β5, and α7, in which several hydrogen bonds were formed by L146-A158 (on the β4-P-loop) and two salt bridges were formed by K192 (on α7). An unexpected and significant change was found in the α1 region, which was greater than 2 standard deviations of the mean of the chemical shift differences, indicating the important conformational regulation of Dat. Consistent with our results, F42-T51 on α1 showed backbone hydrogen bonds changes between the apo-Dat and the Dat/Ac-CoA complex in crystal structures (Supplementary Table 1). The changes in α1 may be induced by Ac-CoA, which switches the structure from the open to the closed form in the β4-α6 region and creates an interhelix interaction between D46 (on α1) and R153 (on the P-loop) (Wu et al., 2020). This result was also similar to that found for AANAT3 from Bombyx mori, in which the change in α1 was important for further formation of the substrate binding site (Aboalroub, Bachman, et al., 2017). Because F42-D46 is conserved among insect AANATs, changes in the conformation of the α1 region in the Ac-CoA-bound state could be an important signature for substrate binding and selection.

The ¹HN, ¹⁵N, ¹³C', ¹³Cα, and ¹³Cβ chemical shifts for the free and Ac-CoA-bound forms of Dat have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under accession numbers 50445 and 28139, respectively.

Author Contribution

P.-C.L., S.-C.S., and S.T. contributed to the conceptualization, methodology, and supervision; C.-Y.W. collected data, wrote the manuscript, and prepared Figures 1–3; I.-C.H. and Y.-Z.L. conducted the investigation and processed the datasets; L.-Y.C. and W.-C.D. prepared the NMR samples; C.-Y.W. and J.W. performed the data analysis. All authors reviewed the manuscript.

Acknowledgement

We express our gratitude for the NMR facility at National Tsing Hua University. This work was supported by the Ministry of Science and Technology, Taiwan (MOST grant numbers 106-2311-B-007-004-MY3 to P.-C.L.)

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Backbone resonance assignments of dopamine N-acetyltransferase in free and cofactor- bound states

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