The YHS-Domain of an Adenylyl Cyclase from Mycobacterium phlei Is a Probable Copper-Sensor Module

Jürgen Ulrich Linder

doi:10.1371/journal.pone.0141843

Abstract

YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn²⁺ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu²⁺ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn²⁺ in the absence of an activator. However, 1–10 μM Cu²⁺ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn²⁺. With Mg²⁺ as the probable physiological cofactor of the adenylyl cyclase Cu²⁺ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg²⁺. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu²⁺ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.

Citation: Linder JU (2015) The YHS-Domain of an Adenylyl Cyclase from Mycobacterium phlei Is a Probable Copper-Sensor Module. PLoS ONE 10(10): e0141843. https://doi.org/10.1371/journal.pone.0141843

Editor: Oleg Y. Dmitriev, University of Saskatchewan, CANADA

Received: July 28, 2015; Accepted: October 13, 2015; Published: October 29, 2015

Copyright: © 2015 Jürgen Ulrich Linder. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The study was made possible by general resources of the University of Bayreuth.

Competing interests: The author has declared that no competing interests exist.

Introduction

Cellular signal transduction is achieved by a complex molecular network to enable the cell to adapt to and react to changes in its environment. Many signal transduction processes involve second messengers. Upon activation of a single second messenger generating protein many of these signaling molecules are produced thereby amplifying the original input signal.

A central second messenger in eukaryotic as well as prokaryotic signal transduction is 3’,5’-cyclic adenosine monophosphate (cAMP). cAMP is generated from ATP by adenylyl cyclases. To date six classes of adenylyl cyclases (ACs) have been described which do not share any sequence similarity and are thought to be the product of convergent evolution [1–4]. By far the largest number of ACs belongs to class III. Class III ACs are found in metazoans including mammals, in protozoans and in eubacteria [1].

The catalytic domain of class III ACs is often termed CHD (cyclase homolgy domain). It forms head-to-tail dimers with the catalytic centers located at the interface of the dimer, thus making dimerization a prerequisite for catalytic activity [5,6]. Based on analysis of amino acid sequences and structures of their catalytic domains the class III ACs have been classified in four categories, class IIIa through class IIId [7]. According to the Interpro protein domain organisation database almost all class III ACs are multi-domain proteins [8]. The general role of many additional domains is that of signal receivers. A stimulus acting on those domains is transmitted to the catalytic domain, usually leading to activation of the CHD [9–12]. Because often regulatory domains associated with ACs are also occuring in other families of signal transduction proteins, the investigation of such ACs can greatly further our understanding of these domains in a more general context [9,12,13].

In the present study we explored the function of the YHS-domain. The YHS-domain is a small cytosolic protein domain of ca. 50 amino acids named after three conserved amino acid residues, i.e. tyrosine, histidine, serine. It has first been published as part of the InterPro database (InterPro IPR007029). The YHS-domain shares extensive similarity to the TRASH domain, a protein domain binding transition-metal ions via conserved cysteine residues [14]. Due to the similarity of YHS to TRASH many domains in InterPro are annotated as both, YHS and TRASH (Fig 1B). Thus, the YHS-domain may be regarded as an expansion of the TRASH domain.

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Fig 1. Sequence analysis of YHS-domains in ACs.

(A) Modular organisation of ACs containing YHS-domains. Amino acid numbering refers to the AC from M. phlei. (B) Alignment of YHS-, YHS/TRASH- and TRASH-domains from various bacterial proteins. The four conserved residues implicated in transition-metal binding are shaded black. Other conserved residues are shaded grey. M. phlei AC, GenBank EID14989.1; Mycobacterium marinum AC, GenBank ACC39874.1; Rhizobium leguminosarum AC, GenBank KEC71354.1; Fulvivirga imtechensis AC, GenBank ELR73472.1; Rhizobium leguminosarum Cu-ATPase, GenBank AAD26860.1; Mesorhizobium loti P-type ATPase, GenBank BAB51797.1; Pseudomonas mendocina toluene-4-hydroxylase, GenBank AAA25999.1; Halovivax ruber permease, GenBank AGB15007.1; Haloarcula marismortui ribosomal protein L24e, GenBank AAV45180.1; Desulfurococcus fermentans YHS-protein, GenBank AFL66382.1.

https://doi.org/10.1371/journal.pone.0141843.g001

Because of its occurrence in a number of bacterial Cu-ATPases it has been proposed that the YHS-domain binds copper ions. However, binding of copper ions to YHS/TRASH has not been experimentally demonstrated in these transporters. A YHS-domain is also found in toluene-4-hydroxylase. The crystal structure of this enzyme has been solved including the YHS-domain [15]. However, neither a metal ion nor any other ligand is bound to YHS in the crystal. Furthermore copper ions have not been implicated in the catalytic mechanism or regulation of the hydroxylase and the function of the YHS-domain is unknown. The prokaryotic ribsomal protein L24e consists of a single TRASH domain. In the crystal structure of the 50S ribosome from H. marismortui [16], a Cd²⁺-ion is bound to L24e via the conserved cysteine residues giving experimental evidence of a transition-metal ion binding TRASH-domain and thus corroborating the proposed function of YHS-domains.

YHS-domains are also present in about 20 putative class III adenylyl cyclases, predicted from whole genome sequencing data. They occur mainly in mycobacterial species, but also in diverse eubacteria like Gordonia bronchialis and several Rhizobium species. In these ACs the CHD is fused to the YHS-domain located at the C-terminal end.

Here we expressed, purified and characterized the YHS-domain containing AC from Mycobacterium phlei. We show that the enzyme is an active AC and is activated by copper ions. Activation relies on the presence of the YHS-domain. Thus the AC-associated YHS-domain appears to act as a sensory module for copper ions.

Materials and Methods

Cloning of Mycobacterium phlei AC

The gene for M. phlei AC (MpAC, GenBank accession EID14989.1) was codon-optimized for expression in E. coli and fitted with an N-terminal BamHI and a C-terminal HindIII site (S1 Fig). The DNA was synthesized commercially (GeneArt/life technologies). For expression of MpAC_1-182 the synthetic gene was transfered via the respective BamHI and HindIII sites into the vector pQE30 (Qiagen). The YHS-domain was then removed by cutting with SphI and HindIII, blunting the ends by treatment with Klenow-enzyme and recircularization of the product. The resulting open reading frame codes for MRGSH₆GS-MpAC_1-182-A. For expression of the full-length enzyme the gene was cloned into the BamHI and HindIII sites of a modified pQE30-vector coding for a TEV-protease recognition sequence downstream of the hexahistidine motif. The open reading frame codes for MRGSH₆GSENLYFQGS-MpAC_1-233 (His-TEV-MpAC_1-233). Expression plasmids were sequenced for checking the intactness of the expression cassettes.

Expression and purification of M. phlei adenylyl cyclase

MpAC was produced in E. coli BL21(DE3)[pRep4]. Batch size was 1 liter of LB-medium supplied with 100 mg/l ampicillin and 25 mg/l kanamycin. Cells were induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside for 5–6 hrs at 20°C. Bacteria were washed with buffer (50 mM Tris/HCl, 1 mM EDTA, pH 8), frozen in liquid nitrogen and stored at -80°C. Purification was started by suspending the cells in 20 ml lysis buffer (50 mM Tris/HCl pH 8), sonicating for 40 s and treating with 0.2 mg/ml lysozyme for 30 min on ice. After addition of 5 mM MgCl₂ and 20 μg/ml of DNaseI the incubation was continued for another 30 min. After centrifugation (31000 x g, 30 min) the solution was supplemented with 250 mM NaCl and 15 mM imidazole (final concentrations). 600 μl Ni-NTA-agarose were added and the mixture was gently shaken for 3 hours on ice. The resin was transferred into a column, washed with 10 ml buffer A (lysis buffer containing 250 mM NaCl, 15 mM imidazole, 5 mM MgCl₂) and subsequently with 5 ml of buffer B (lysis buffer containing 15 mM imidazole, 5 mM MgCl₂). Protein was eluted with 0.6 ml of buffer C (37.5 mM Tris/HCl, pH 8, 250 mM imidazole, 2 mM MgCl₂). Purified MpAC was stored at -20°C after addition of 40% glycerol. The purity of the recombinant proteins was assessed by 14% SDS-PAGE and staining with Coomassie Blue G250.

Removal of the hexahistidine-tag of full-length MpAC

Purified His-TEV-MpAC_1-233 was diluted with an equal volume of 20 mM Tris/HCl, pH 8 and 0.12 μg His-tagged TEV-protease per μg AC was added. The mixture was incubated for 12 hours at 4°C. Buffer was rapidly changed by consecutive dilution and ultrafiltration to 20 mM Tris/HCl, pH8; 7 mM Imidazole; 50 mM NaCl; 2 mM MgCl₂. 250 μl Ni-NTA-Agarose per 100 μg of protein were added and incubation was performed for 3 hours on ice. The resin was removed by filtration. The filtrate was mixed with 25% glycerol and stored at -20°C. The final product carries an N-terminal dipeptide compared to native MpAC. The sequence is GS-MpAC_1-233.

AC assay

AC activity was measured for 10 min at 30°C in a volume of 30 μl. Standard reactions contained 2.5 mM Tris/HCl, pH 8.0, 5 mM MnCl₂ or MgCl₂, 1 mM ATP. cAMP was determined by HPLC as described previously [17]. All data are means of 2 to 6 points and are denoted with their standard deviations.

Tryptophane-fluorescence spectroscopy

Samples were irradiated in a 1 ml fluorescence cuvette at 295 nm (gap width 5 nm) at 20°C. Fluorescence intensities at 345 nm were recorded with a gap width of 5 nm. Fluorescence data were corrected for the inner-filter effect of protein and reagents and for the dilution factor upon addition of reagents.

Results and Discussion

Primary structure of ACs containing a YHS domain

A search of the InterPro protein domain architecture database [8] for protein sequences containing class III adenylyl cyclase catalytic domains (CHDs, InterPro IPR001054) yielded a list of 1369 different architectures present in a total of 19961 protein sequences. Among these, 13 proteins were found which contained a YHS domain. All share the same architecture of a single CHD linked to a single C-terminal YHS domain (Fig 1A). The linker region between the two domains consists of 8–11 residues. Using the AC-YHS protein from Mycobacterium phlei (GenBank accession EID14989.1) as a query, a BLAST search of the non-redundant protein data base [18] yielded a set of 24 sequences, partially overlapping the results from the InterPro search. A total number of 26 putative AC-YHS enzymes were detected by the combination of both searches. A representative sequence alignment is given in the supplementary material (S2 Fig).

We have focused our work on the AC form M. phlei because of its compact structure with 233 residues compared to a range of 216–473 amino acids (aa) among all AC-YHS sequences and because M. phlei is a well characterized species with a history as a mycobacterial model organism [19]. Inspection of the CHD of the M. phlei AC revealed that all six canonical catalytic residues [5,7] are present (highlighted in S2 Fig). Furthermore the dimerization arm of the M. phlei AC is short with 7 residues, which classifies it as a class IIIc CHD [7]. Analysis of the complete set of AC-YHS proteins shows that all of them belong to that subclass.

An alignment of the YHS domains of ACs with the YHS/TRASH domains of Cu-ATPases and other YHS and TRASH domains is shown in Fig 1B. The mode of metal-ion coordination has previously been uncovered by the crystal structure of the ribosomal TRASH-domain subunit L24e [16]. A Cd²⁺ ion is bound to four cysteine residues which are highlighted in Fig 1B. In the YHS domain of ACs the N-terminal cysteine is replaced by aspartate (D178 in M. phlei AC), while the other three cysteine residues are conserved (M. phlei AC: C181, C204, C208). An aspartate in the position of the first metal-ion coordinating residue appears to be common among YHS and TRASH-domains (Fig 1B). In general the sequence profile of the YHS-domains of bacterial ACs fits that of bacterial Cu-ATPases and other metal-ion binding YHS- and TRASH-domains, which led us to the hypothesis that the AC of M. phlei may bind transition-metal ions, probably copper, via its YHS-domain and that it may be regulated by such ions.

Expression and characterization of the catalytic domain of M. phlei adenylyl cyclase

In a first step towards investigation of the YHS-domain the isolated catalytic domain of M. phlei AC (MpAC) was characterized to provide a base for the separation of direct effects of metal-ions on the CHD from effects mediated by the YHS-domain. The CHD (aa 1–182) of MpAC was N-terminally tagged with a hexahistidine motif and expressed in E. coli. Upon purification of MpAC_1-182 by affinity chromatography the protein migrated at 22 kDa on SDS-PAGE (calculated 20.1 kDa, Fig 2A). The identity of the protein was confirmed by mass spectrometry of a tryptic digest (data not shown). MpAC_1-182 showed robust AC activity of 196 ± 12 nmol cAMP mg^-1 · min^-1 with Mn²⁺ as a cofactor whereas activity with Mg2+ was minimal with 0.37 ± 0.02 nmol cAMP mg^-1 · min^-1. With Mn²⁺ as a cofactor MpAC_1-182 displayed maximal activity in the range of pH 8.0–8.5. The temperature optimum was at 30°C (data not shown). Subsequently all assays were performed at 30°C and pH 8.0.

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Fig 2. Biochemical analysis of the catalytic domain MpAC_1-182.

(A) SDS-PAGE of MpAC_1-182. (B) Substrate kinetics measured with 5 mM Mn²⁺ as a cofactor at 30°C and pH 8.0. Vertical bars indicate the standard deviation if larger than symbol size. (C) Mn²⁺-dependance assayed at 1 mM ATP. (D) Inhibition by Cu²⁺. Reactions contained 1.6 μM MpAC_1-182, 5 mM Mn²⁺ and 1 mM ATP.

https://doi.org/10.1371/journal.pone.0141843.g002

Kinetic analysis showed a v_max of 335 ± 70 nmol cAMP mg^-1 · min^-1 with half-maximal activity (SC₅₀) at 0.90 ± 0.22 mM ATP and a pronounced positive cooperativity for ATP as seen by a Hill coefficient of 1.5 ± 0.2 (Fig 2B, Table 1). The Mn²⁺-dependence of MpAC_1-182 at 1 mM ATP is shown in Fig 2C. Activity is low up to 1 mM Mn²⁺ and surging when the concentration of Mn²⁺ exceeds that of ATP. This behavior is typical for the two-metal ion mechanism of ACs and demonstrates the requirement of both, MnATP and free Mn²⁺ for catalysis to occur [20]. Based on a reciprocal plot the affinity of MpAC_1-182 for the cofactor was calculated yielding an EC₅₀ for free Mn²⁺ of 2.0 ± 0.6 mM.

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Table 1. Kinetic parameters of MpAC_1-182 and MpAC_1-233.

https://doi.org/10.1371/journal.pone.0141843.t001

Because copper ions are activators of full-length MpAC (see below), the effect of Cu²⁺ on MpAC_1-182 was tested (Fig 2D). Cu²⁺ acted as an inhibitor of the CHD with an IC₅₀ of 20 μM.

Expression and characterization of full-length M. phlei adenylyl cyclase

The full-length MpAC was fitted with an N-terminal hexahistidine-tag carrying a TEV-protease recognition sequence and expressed in E. coli. Purified His-TEV-MpAC_1-233 appeared at 31 kDa on SDS-PAGE, slightly higher than expected (calculated 27.0 kDa, Fig 3A). Subsequently the hexahistidine-tag was removed with TEV protease and the untagged full-length M. phlei AC was purified by reverse Ni²⁺-affinity chromatography. Untagged MpAC_1-233 showed a slightly higher mobility on SDS-PAGE compared to His-TEV-MpAC_1-233 demonstrating the success of the tag-removal procedure (Fig 3A).

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Fig 3. Biochemical analysis of full-length MpAC_1-233.

(A) SDS-PAGE of His-TEV-MpAC_1-233 (+tag) and untagged MpAC_1-233 (-tag). Both lanes are from the same gel and are shown in alignment with the molecular weight marker on that gel. (B) Substrate kinetics measured with 5 mM Mn²⁺ as a cofactor at 30°C and pH 8.0. (C) Mn²⁺-dependance assayed at 1 mM ATP.

https://doi.org/10.1371/journal.pone.0141843.g003

The specific activity of MpAC_1-233 with Mn²⁺ as a cofactor was 541 ± 56 nmol cAMP mg^-1 · min^-1, while no activity was detectable with Mg²⁺. The v_max of 1660 ± 90 nmol cAMP mg^-1 · min^-1 and SC₅₀ of 2.11 ± 0.54 mM ATP were higher than the corresponding values of the isolated CHD (Fig 3B, Table 1). In addition, MpAC_1-233 did not show cooperativity with a Hill coefficient of 0.9 ± 0.1. The Mn²⁺-dependance of MpAC_1-233 showed a similar requirement for Mn²⁺ in excess of ATP like that of MpAC_1-182. Yet, the affinity for Mn²⁺ was lower for MpAC_1-233 with an EC₅₀ of 8.3 ± 0.6 mM free Mn²⁺ (Fig 3C). The change in the kinetic parameters and the Mn²⁺-affinity by the presence of the YHS-domain indicates that the YHS-domain has an impact on the conformation of the CHD even in the absence of transition-metal ions.

Stimulation of full-length M. phlei adenylyl cyclase by copper ions with Mn²⁺ as a cofactor

The hypothesis of the YHS-domain of M. phlei AC being a transition-metal ion binding domain with similarity to the YHS-domains of CuATPases was tested in assays of the untagged full-length MpAC_1-233 with micromolar concentrations of Cu²⁺ and Mn²⁺ as a cofactor (Fig 4A). Cu²⁺ maximally stimulated MpAC_1-233 sixfold at concentrations of 1 to 10 μM. Because we used an enzyme concentrations of 0.4 μM to assure efficient dimerization of MpAC_1-233 (see below), assays with Cu²⁺ below 1 μM were not conducted. Yet, the data imply that the EC₅₀ for activation by Cu²⁺ is in the nanomolar range. On the other hand, high concentrations of Cu²⁺ were inhibitory similar to the results seen with the isolated catalytic domain. Thus, we concluded that the stimulatory effect of low concentrations of Cu²⁺ on MpAC_1-233 is mediated by the YHS-domain. The inhibitory effect of higher concentrations of Cu²⁺ may be due to general interactions and reactions with protein similar to those exploited in classic protein assays like the Lowry- and the Biuret-method [21]. Kinetic analysis of MpAC_1-233 showed that 10 μM Cu²⁺ led to an increase in v_max and a concomitant decrease of SC₅₀ for ATP (Fig 4B, Table 1). The Hill coefficient of 1.2 ± 0.3 indicated that Cu²⁺ did not induce pronounced cooperativity for ATP. Furthermore, addition of 10μM Cu²⁺ resulted in a 10-fold higher affinity for the cofactor Mn²⁺ with an EC₅₀ of 0.7 ± 0.2 mM for the free ion (Fig 4C). Taken together Cu²⁺ appears to increase the catalytic efficiency of the enzyme.

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Fig 4. Stimulation of full-length MpAC_1-233 by Cu²⁺ assayed with Mn²⁺ as a cofactor.

(A) Effect of Cu²⁺. Reactions contained 0.4 μM MpAC_1-233, 5 mM Mn²⁺ and 1 mM ATP. (B) Substrate kinetics measured with 5 mM Mn²⁺ as a cofactor and 10 μM Cu²⁺. (C) Mn²⁺-dependance assayed at 1 mM ATP and 10 μM Cu²⁺.

https://doi.org/10.1371/journal.pone.0141843.g004

Activation of full-length M. phlei adenylyl cyclase by copper ions with Mg²⁺ as a cofactor

Because a requirement of millimolar concentrations of Mn²⁺ for AC activity may not reflect the physiological environment of the enzyme in Mycobacterium phlei we explored the effect of Cu²⁺ on MpAC_1-233 with Mg²⁺ as a cofactor. MpAC_1-233 produced 122 ± 20 nmol cAMP mg^-1 · min^-1 upon addition of 10 μM Cu²⁺. Thus Cu²⁺ served as an on-switch of MpAC that relies on the YHS-domain of the enzyme.

Kinetic analysis (Fig 5A) yielded a v_max of 485 ± 35 nmol cAMP mg^-1 · min^-1 with an SC₅₀ of 1.63 ± 0.23 mM ATP. The Hill coefficient of 1.5 ± 0.1 demonstrated a positive cooperativity for ATP with Mg²⁺ in contrast to assays with Mn²⁺ (Table 1). Mg²⁺-dependance showed the typical characteristics of a two-metal ion mechanism as seen with Mn²⁺; EC₅₀ for free Mg²⁺ was 1.8 ± 0.8 mM (Fig 5B). Activation by Cu²⁺ was maximal in the range of 1 to 10 μM with strong inhibition at higher concentrations (Fig 5C).

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Fig 5. Activation of full-length MpAC_1-233 by Cu²⁺ assayed with Mg²⁺ as a cofactor.

(A) Substrate kinetics measured with 5 mM Mg²⁺ as a cofactor and 10 μM Cu²⁺. (B) Mg²⁺-dependance assayed at 1 mM ATP and 10 μM Cu²⁺. (C) Effect of Cu²⁺. Reactions contained 0.4 μM MpAC_1-233, 5 mM Mg²⁺ and 1 mM ATP. (D) Tryptophane-fluorescence spectroscopy. The fluorescence emission signal at 345 nm of 1.15–2.55 μM MpAC_1-233 (n = 3) or 5 μM N-acetyl-tryptophaneamide (NATA, n = 2) was taken as 100% (-). Subsequently 25 μM Cu²⁺ were added (Cu²⁺) and finally the copper-ions were complexed with 375 μM EDTA (EDTA).

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If the YHS-domain served as a Cu²⁺-sensor the activation of MpAC_1-233 should be specific to copper ions. MpAC_1-233 was assayed with Mg²⁺ as a cofactor and addition of Zn²⁺, Fe²⁺, Fe³⁺, Cr³⁺, Co²⁺ and Ni²⁺. There was no detectable AC activity with any of these transition-metal ions. Next we checked whether activation of MpAC_1-233 by Cu²⁺ occurs by binding of the metal ion to the enzyme or whether it is a consequence of the oxidative properties of Cu²⁺, e.g. by oxidizing the conserved cysteine residues [22] of the YHS-domain. MpAC_1-233 was incubated with 10 μM Cu²⁺ for 15 min in the presence of Mg²⁺ as a cofactor, then Cu²⁺ was selectively quenched by 375 μM EDTA before the reaction was started by addition of ATP. No AC activity was detected, while in a parallel experiment under same conditions, but without quenching by EDTA, AC activity was preserved (data not shown).

Finally, we investigated the effect of Cu²⁺ on the YHS-domain by tryptophane-fluorescence spectroscopy, which was facilitated by the only two tryptophane residues of MpAC_1-233 being located there (W201 and W202). The fluorescence emission spectrum showed a maximum at 345 nm, which was unchanged by the addition of Cu²⁺ or EDTA (data not shown). However, the fluorescence intensity at 345 nm was significantly reduced by Cu²⁺ and addition of EDTA in essence reverted the effect (Fig 5D). As a control, the fluorescence of N-acetyl-tryptophanamide was unaffected by Cu²⁺ and EDTA showing that neither reagent acted as a general quencher of fluorescence (Fig 5D). The data are consistent with a conformational change of the YHS-domain triggered by the binding of Cu²⁺. Thus we favor the view that the M. phlei AC acts as a Cu²⁺-sensor enzyme, where binding of Cu²⁺ to the YHS-domain in turn activates the CHD.

Although we do not know the mechanism of activation by Cu²⁺ at this time, we can exclude an enhanced dimerization as an underlying principle. The dissociation constant of the enzyme is 0.16 μM as calculated from the protein dependance of the AC activity (data not shown). We used at least 0.4 μM MpAC_1-233 in all assays to ensure that the enzyme largely remains dimerized. Crystal structures of class III ACs have shown that upon activation only minor changes in secondary structure occur, whereas often pronounced shifts in the orientation of the CHD monomers to each other are visible [5,10,17]. Similar mechanisms might trigger the activation of the M. phlei ACby Cu²⁺.

The physiological role of the Cu²⁺-sensor AC in the bacterium remains open to speculation. It has been known since the 1950’s that M. phlei is exquisitely sensitive to Cu²⁺ with strong growth inhibition occurring with as little as 1 μM Cu²⁺ in the medium [23]. For comparison 100 μM Cu²⁺ has no effect on the growth rate of E. coli [23]. A link between Cu²⁺-sensitivity and Cu²⁺-sensor AC in M. phlei therefore appears as a plausible hypothesis.

By structural analogy to the related TRASH-domain the conserved aspartate and cysteine residues marked in Fig 1B are likely to coordinate Cu²⁺ in the YHS-domain of M. phlei AC [14]. Our study provides biochemical evidence for a function of the YHS-domain in transition-metal ion binding and enzyme regulation which also impacts on our understanding of the TRASH-domain. Furthermore our data underline the high versatility of class III AC catalytic domains in their interaction with diverse regulatory domains as seen in past studies.

Supporting Information

S1 Fig. Sequence of the synthetic gene for Mycobacterium phlei AC.

Flanking BamHI and HindIII are shown in italics. Differences to the natural gene (GenBank EID14989.1) are underlined.

https://doi.org/10.1371/journal.pone.0141843.s001

(TIF)

S2 Fig. Alignment of ACs carrying a C-terminal YHS-domain.

In the CHD, critical functional residues for metal-cofactor binding (M), adenine-moiety binding (A) and for catalysis (C) are shaded black, as well as the four conserved putative transition-metal ion binding residues of the YHS-domain. Sequences are from Mycobacterium phlei (GenBank EID14989.1), Mycobacterium marinum (GenBank ACC39874.1), Williamsia sp.ARP1 (NCBI reference WP_045825068.1); Candidatus Microthrix parvicella RN1; (GenBank CCM65060.1); Rhizobium leguminosarum (GenBank KEC71354.1); Rhizobium etli (NCBI reference WP_040141435.1); Fulvivirga imtechensis (GenBank ELR73472.1).

https://doi.org/10.1371/journal.pone.0141843.s002

(TIF)

Acknowledgments

The author thanks Julian Pfahler and Elena Müller for performing initial experiments. The research project was funded by the University of Bayreuth.

Author Contributions

Conceived and designed the experiments: JUL. Performed the experiments: JUL. Analyzed the data: JUL. Contributed reagents/materials/analysis tools: JUL. Wrote the paper: JUL.

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Figures

Abstract

Introduction

Materials and Methods

Cloning of Mycobacterium phlei AC

Expression and purification of M. phlei adenylyl cyclase

Removal of the hexahistidine-tag of full-length MpAC

AC assay

Tryptophane-fluorescence spectroscopy

Results and Discussion

Primary structure of ACs containing a YHS domain

Expression and characterization of the catalytic domain of M. phlei adenylyl cyclase

Expression and characterization of full-length M. phlei adenylyl cyclase

Stimulation of full-length M. phlei adenylyl cyclase by copper ions with Mn2+ as a cofactor

Activation of full-length M. phlei adenylyl cyclase by copper ions with Mg2+ as a cofactor

Supporting Information

S1 Fig. Sequence of the synthetic gene for Mycobacterium phlei AC.

S2 Fig. Alignment of ACs carrying a C-terminal YHS-domain.

Acknowledgments

Author Contributions

References

Stimulation of full-length M. phlei adenylyl cyclase by copper ions with Mn²⁺ as a cofactor

Activation of full-length M. phlei adenylyl cyclase by copper ions with Mg²⁺ as a cofactor