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Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity

BMC Biochemistry volume 12, Article number: 35 (2011) Cite this article

Abstract

Background

Methionine aminopeptidase (MetAP) is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins.

Results

We cloned genes encoding two such metalloproteases (Mt MetAP1a and Mt MetAP1c) present in Mycobacterium tuberculosis and expressed them as histidine-tagged proteins in Escherichia coli. Although they have different substrate preferences, for Met-Ala-Ser, we found, Mt MetAP1c had significantly high enzyme turnover rate as opposed to Mt MetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50°C) of Mt MetAP1a compared to that of the Mt MetAP1c. Modelling of Mt MetAP1a based on Mt MetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of Mt MetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of Mt MetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of Mt MetAP1c.

Conclusion

Our findings unequivocally emphasized that N-terminal extension of Mt MetAP1c contributes towards the functionality of the enzyme presumably by regulating active site residues through "action-at-a-distance" mechanism and we for the first time are reporting this unique function of the enzyme.

Background

The N-terminal methionine excision (NME) is an essential co-translational proteolytic process responsible for the diversity of amino-termini of proteins in both prokaryotes and eukaryotes [1]. It is an irreversible reaction, which occurs soon after N-terminal residues of the nascent polypeptide chain emerge from the ribosome exit tunnel before the commencement of protein folding [2]. The enzyme involved in the process is known as methionine aminopeptidase (MetAP), which specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged [3]. In prokaryotes and eukaryotic organelles where the initiator methionine is formylated, NME requires the action of another metalloprotease, peptide deformylase in addition to MetAP, which removes the N-formyl group present on all synthesized nascent polypeptides. In fact, removal of N-formyl group is a prerequisite for the subsequent action of MetAP [4]. Nevertheless, the MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins [5].

MetAPs are ubiquitous in distribution and are highly conserved throughout the phylogeny. They were initially grouped in two classes, MetAP1 and MetAP2 on the basis of sequence comparison. The type 2 enzymes, in contrast to type 1, have an approximately 60 amino acid long α helical domain inserted within the catalytic region of the enzyme [6, 7]. This helical sub-domain shares neither sequence nor structural homology with any other known protein. The MetAPs are further divided in two subclasses on the basis of absence (subclass a) or presence (subclass b) of N-terminal extension [8]. The N-terminal extension present in type 1b contains two zinc finger motifs while that of type 2b has alternate stretches of polyacidic and polybasic residues. Recently, two new subclasses, type 1c and 2c, have been introduced where ~40 amino acid long N-terminal extension is present but zinc finger motif is absent [9, 10].

Apart from several types of MetAPs, presence of more than one copy of functional genes encoding this enzyme has been reported in both prokaryotes and eukaryotes. In human, there are two such well characterized enzymes, which have been shown to be involved in cell proliferation [11, 12]. Among four isoforms of MetAPs in Plasmodium falciparum, three exhibited enzymatic activities [13]. Genetic studies further established the essentiality of this enzyme in both prokaryotes and eukaryotes [14–16]. These findings led to the assumption that MetAP would be an ideal drug target and screening/designing of inhibitors against this enzyme might help in planning to combat the problem of rapid resurgence of diseases due to emergence of drug resistant strains of different pathogens. In this context, we focussed on tuberculosis, which is caused by Mycobacterium tuberculosis, and responsible for considerable human mortality throughout the world in recent years.

Although bacteria generally have single MetAP1, mycobacterial genome revealed the presence of 2-4 putative genes for MetAP1; for instance M. tuberculosis has two genes mapA (Rv0734) and mapB (Rv2861c) encoding Mt MetAP1a and Mt MetAP1c respectively. Although, crystal structure of Mt MetAP1c was solved as an apoenzyme and in complex form [9], biochemical characterization of the two Mt MetAPs has not been reported until very recently [17–21]. However, detailed structure-function analysis of these enzymes has not yet been carried out. In this scenario, we concentrated on both these Mt MetAP1s and in this article we report their distinct characteristics. Furthermore, we provide here evidence that catalytic domain of Mt MetAP1c alone is not sufficient for the enzyme activity.

Results

Mt MetAP1a and Mt MetAP1c exhibit subtle difference in their behaviour

The mapA and mapB genes from M. tuberculosis were PCR amplified, cloned in pET-28c and expressed in E. coli strain BL21(DE3). The recombinant N-terminal His-tagged proteins were purified to near homogeneity by immobilized metal affinity chromatography using Ni-NTA resin. The purified proteins, on resolving in SDS-PAGE when visualized in Coomassie Brilliant Blue stained gels, exhibited bands at 37.3 ± 1.7 kDa (n = 4) and 39.1 ± 1.2 kDa (n = 4) for Mt MetAP1a and Mt MetAP1c, respectively. The authenticity of the expressed proteins was further confirmed by Western blotting using anti-His antibody (data not shown). Mt MetAP1a and Mt MetAP1c displayed maximum activity with Met-Gly-Met-Met and Met-Ala-Ser respectively. The enzymatic specificity for the terminal methionine residue was confirmed by using Gly-Gly-Ala as the substrate and as expected both the enzymes were unable to hydrolyze it (Figure 1A). Furthermore, using different peptides as substrates revealed that at least a tripeptide is the prerequisite for their functionality, since both the enzymes showed almost negligible activity with the dipeptide Met-Gly.

Figure 1
figure 1

Mt MetAP1a and Mt MetAP1c are enzymatically active. (A) Methionine aminopeptidase activity with different substrates. Enzyme activity of the two methionine aminopeptidases was determined using different substrates (4 mM) as indicated and Mt MetAP1a (1.7 nM) or Mt MetAP1c (0.038 nM) of purified protein as mentioned in the methods. Notations used: MG, Met-Gly; MAS, Met-Ala-Ser; MGMM, Met-Gly-Met-Met; GGA, Gly-Gly-Ala. (B) Kinetic analysis of methionine aminopeptidase activity of Mt MetAP1s. Kinetic analysis of methionine removal ability of Mt MetAP1a (left) was carried out by using 1.7 nM of purified protein with increasing concentration of Met-Gly-Met-Met as substrate. The reaction was monitored for 15 min. 0.038 nM of purified protein was incubated for 5 min with increasing concentration of Met-Ala-Ser as substrate to determine the kinetic parameters of Mt MetAP1c (Right). Insets, effect of increasing amount of protein on the enzyme activity. (C) Effect of different inhibitors and chelator on Mt MetAPs activity. MetAP1s were preincubated for 15 min at room temperature with the indicated amount of the inhibitors/EDTA and then the activity assay was performed. (D) Effect of temperature on Mt MetAP1s. The thermal unfolding was performed at the rate of 1°C/min and the change in the CD signal was monitored at 208 nm. Left and right panels represent the thermal denaturation graphs for Mt MetAP1a and Mt MetAP1c respectively. Upper insets show the absorption spectra of Mt MetAP1a and Mt MetAP1c monitored at 25°C and 50°C. Lower insets show the effect of preincubation for 10 min at indicated temperatures on the activity of two Mt MetAP1s. Mt MetAP1a and Mt MetAP1c are depicted at (left) and (right) respectively. The assay was carried out at 30°C after preincubation.

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Activities of purified enzymes (1.7 nM for Mt MetAP1a or 0.038 nM for Mt MetAP1c) as a function of increasing concentrations (0.75-8 mM) of substrate (Met-Gly-Met-Met for Mt MetAP1a or Met-Ala-Ser for Mt MetAP1c) yielded typical Michaelis-Menten curves (Figure 1B). Interestingly, kinetic parameters determined by fitting the data to Michaelis-Menten equation for both the enzymes for their preferred substrate indicated that they were active (Table 1; see Km and kcat values with Met-Gly-Met-Met for Mt MetAP1a and Met-Ala-Ser for Mt MetAP1c; considering recombinant His-tagged proteins). Interestingly, using Met-Ala-Ser as the substrate for both the enzymes the catalytic efficiency of Mt MetAP1c was strikingly high (~350-fold) compared to that of the Mt MetAP1a (Table 1; kcat/Km = ~3 mM-1s-1 as opposed to ~1156 mM-1s-1). Thus, our result is in consonance with a recent report that Mt MetAP1c is catalytically more efficient compared to Mt MetAP1a [20]. Expectedly, both the enzymes exhibited a linear increase in activity as a function of increasing protein concentrations (r = 0.997 or 0.991; Figure 1B, insets at both left and right panels).

Table 1 Kinetic parameters of Mt MetAPs
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Since MetAPs are known as dinuclear metalloproteases, we also monitored the mycobacterial enzyme activities in response to EDTA, a metal ion chelator. As expected, metal ion chelator affected the activities of both the enzymes. However, compared to that used for Mt MetAP1c enzyme activity of Mt MetAP1a was inhibited at a lower concentrations of EDTA (Figure 1C). Leucine aminopeptidase inhibitors like amastatin and bestatin, have already been shown to inhibit MetAP activity by binding to its metal centre [7]. As shown in Figure 1C, amastatin affected both Mt MetAP1a and Mt MetAP1c enzyme activities; magnitude of inhibition, however, varied considerably between them (compared to untreated control, ~80% decrease in Mt MetAP1a as opposed to ~45% in Mt MetAP1c with 100 μM of amastatin). Bestatin, on the other hand, had no effect on the enzymatic activities of both the proteins within the concentration range tested in our experimental conditions.

We further addressed the adaptability of Mt MetAP1a and Mt MetAP1c enzymes to different temperatures. For this purpose, both the enzymes following incubation at different temperatures (4° or 30° or 37° or 50°C for 10 min) were assayed at 30°C for their activities. The maximum activity for both the enzymes was noticed between 30°-37°C. Interestingly, Mt MetAP1a retained ~85% of its activity at 50°C as opposed to ~25% by the Mt MetAP1c compared to that observed at 30°C (insets showing enzyme activities in left and right panels of Figure 1D). This observation led to the postulation that alteration in the enzyme activity might be occurring as a result of heating and this would very likely cause structural changes in both the proteins. This possibility was explored by incubating both the proteins to 50°C and comparing their far-UV CD spectra between 190-250 nm. Mt MetAP1a retained (> 50%) of its secondary structure on heating at 50°C while Mt MetAP1c was unfolded (compare insets showing CD spectrum in left and right panels of Figure 1D). In fact, determination of Tm of both the proteins by CD spectroscopy yielded values of 53.6°C for Mt MetAP1a and 42.7°C for Mt MetAP1c (Figure 1D). These findings, therefore, established the difference in the adaptive ability of Mt MetAP1a to high temperature compared to that of the Mt MetAP1c and bring an end to the argument about the temperature tolerance of the Mt MetAP1s [17, 20]. Thus, all these lines of evidence implicated the existence of subtle differences in the behaviour of two mycobacterial MetAPs.

Contribution of active site residues on Mt MetAP1 enzyme activity

Nucleotide derived amino acid sequence alignment of Mt MetAP1a with Mt MetAP1c revealed ~41% identity (Figure 2A) and the crystal structure of Mt MetAP1c (PDB ID: 1YJ3) has already been resolved. Since Mt MetAP1a did not show any significant identity with any other protein in the PDB database, we used Mt MetAP1c as the template for modelling the structure of this metalloprotease [9]. The modelled structure of Mt MetAP1a was validated by PROCHECK and it shows that 98% residues are in the most favoured regions of Ramachandran plot. The monomer of each protein consists of two pairs of α-helices (α1 to α4) packed on the either side of the central antiparallel β-sheet (β3 and β4). Interestingly, ~55% of the structure comprised of loops connecting the secondary structural elements. However, the 40 amino acid long N-terminal extension of Mt MetAP1c, which is devoid of any secondary structural element, lies across the surface of its catalytic domain (Figure 2B).

Figure 2
figure 2

Mutations of active site residues affect enzymatic activity of Mt MetAPs. (A) Sequence alignment of the two mycobacterial methionine aminopeptidases was performed using Clustal X. Gaps in the sequences were introduced for optimum alignment. Asterisk and dots denote, identical and similar amino acids, respectively. Residues highlighted with black represent the 40 amino acid long N-terminal extension present in Mt MetAP1c and those with gray are the mutated amino acids in the two Mt MetAPs. (B) Structural alignment of Mt MetAP1a (green) with respect to Mt MetAP1c (pink). Residues in green (sticks) represent Mt MetAP1a active site residues (His-88, His-193, Glu-219) and residues in pink (sticks) depict Mt MetAP1c active site residues (His212, His-114, Glu-238). Residue in blue is Tyr-183 (Mt MetAP1a) and in hot pink is Phe-202 (Mt MetAP1c). (C) Methionine aminopeptidase activity of different point mutants. Enzyme activity for the indicated amount of wild-type and purified variants of two Mt MetAP1s was monitored using 4 mM of substrate, Met-Gly-Met-Met (Mt MetAP1a) and Met-Ala-Ser (Mt MetAP1c). Insets, Western blot of mutant proteins using anti-His antibody, far-UV and near-UV CD spectra.

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Structural analysis predicted the presence of His-88, His-193 and Glu-219 in the active site of Mt MetAP1a, while residues like Asp-106, Asp-117, Glu-250 and Trp-236 are lining the perimeter of the active site. The presence of homologous residues in Mt MetAP1c implies that the active site of both the proteins is essentially conserved (Figure 2A). To gain an insight on their contribution towards the enzyme activity, we generated several point mutants (H88A, H193A, D117A, E219V, W236L for Mt MetAP1a and H114A, H212A, D131A, E238A, W255L for Mt MetAP1c). Interestingly, the variants do not show any appreciable enzymatic activity even on using 10-fold excess protein compared to that of the wild-type (Figure 2C). In fact, mutating W236L in Mt MetAP1a and W255L in Mt MetAP1c led to complete loss in the activity (Figure 2C), which is in contrast to the report available with MetAP1 of E. coli[22]. Expression of all the variants, however, was authenticated by Western blotting using anti-His antibody (insets, Figure 2C). To examine the effect of mutations on the secondary and tertiary structures of the Mt MetAP1s, the CD spectral studies were performed. Far-UV CD spectrum of the mutant proteins of Mt MetAP1a revealed that H88A, D117A, E219V and W236L show reduction in helical content. The trend of the data in the region of 200-210 nm suggests that the band with negative mean residue ellipticity at lower wavelengths has reduced signal strength presumably owing to the conversion of some helical regions to less ordered structures (Figure 2C, left panel, inset). Interestingly, mutant H193A shows greater helicity than the wild-type protein. Far-UV CD spectral studies of wild-type Mt MetAP1c and its variants revealed that in D131A although there is no significant reduction in the mean residue ellipticity at the 200-210 nm range, it decreased at the 220-225 nm range (Figure 2C, right panel, inset). On the other hand, in H114A and E238A reduction in band intensity was observed at both 200-210 nm and 220-225 nm. In H212A and W255L the band intensities at the 200-210 nm as well as in 220-225 nm were further reduced (Figure 2C, right panel, inset). Thus, the mutations affected secondary structure of both the proteins and changes observed in the near-UV CD spectra indicated alteration in their aromatic environment (insets of Figure 2C, see both left and right panels).

Role of N-terminal extension of Mt MetAP1c on the enzyme activity

The presence of 40 amino acid N-terminal extension although not unusual among MetAP1s, its contribution towards the enzymatic activity is not known as yet [23–25]. To elucidate this aspect, a series of N-terminal deletion variants for Mt MetAP1c were generated (Figure 3A) and their enzyme activity was monitored (Figure 3B). Surprisingly, compared to the wild-type, deletion constructs (Δ2-10, Δ2-15, Δ2-20, Δ2-30 and Δ2-40) displayed either drastic decrease or no enzymatic activity (Figure 3B). Even use of 10 fold higher amounts of proteins in our assay, Δ2-10 or Δ2-15 exhibited only 30% activity while Δ2-20 or Δ2-30 or Δ2-40 had no activity (Figure 3B). Thus, our results argue that the catalytic domain alone is not sufficient for the enzymatic function of Mt MetAP1c.

Figure 3
figure 3

N-terminal deletion of Mt MetAP1c affects its enzyme activity. (A) Deletion scheme. Notations used: WT, wild-type; Δ, deleted residues. (B) Assessment of methionine aminopeptidase activity of deletion variants. Methionine aminopeptidase activity assay was performed using indicated amount of wild-type and mutant proteins with 4 mM of Met-Ala-Ser as the substrate. (C) Coomassie stained SDS-PAGE gel (upper panel) and Western blot (lower panel) using anti- His antibody of the mutant proteins as shown in Figure 3B. (D) Far-UV CD spectra of the WT, Δ2-10 and Δ2-15 proteins. (E) Near-UV CD spectra of the WT, Δ2-10 and Δ2-15 proteins.

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Mt MetAP1c variants, like Δ2-20 or Δ2-30 or Δ2-40, which did not show any enzyme activity, when stained with Coomassie Brilliant Blue after resolving in SDS-PAGE, displayed a progressive loss in the purity of the proteins with the increase in the length of deleted regions (Figure 3C, upper panel). The authenticity of these mutant proteins was confirmed by Western blotting using anti-His antibody (Figure 3C, lower panel). The effect of mutation was evident by the presence of contaminating bands in the Ni-NTA purified samples (Figure 3C, upper panel) and even reduced protein yield (~4 mg for Δ2-20/Δ2-30/Δ2-40 as opposed to ~9 mg in wild-type average yield from 1 litre culture of E. coli cells). Analysis of its crystal structure revealed that the N-terminal extension present in Mt MetAP1c wraps around the surface of its catalytic domain in contiguous crevice [9]. Hence, its truncation as a result of deletion might have exposed the hydrophobic residues and thereby destabilized the protein. This has been manifested presumably by exhibiting altered proteolytic property of Mt MetAP1c variants and/or led to acquiring the tendency to associate with other proteins [9].

Interestingly, reduction in the enzymatic activity was also observed in Δ2-10 and Δ2-15 variants despite the fact that the Ni-NTA purified proteins appear to be similar in profile as has been observed with wild-type. The far-UV CD spectra showed that they (Δ2-10 and Δ2-15) are folded like the wild-type protein (Figure 3D). A slight alteration observed in the near-UV CD spectra for the Δ2-15 variant (Figure 3E) was very likely the result of alteration in the micro-environment of Trp present at the amino acid position 20. Nonetheless, it is apparent from our results that the first 15 amino acids of Mt MetAP1c are important for the catalytic activity of the enzyme.

Role of Val-18 and Pro-19 on Mt MetAP1c enzyme activity

To elucidate, the presence of any conserved residues between amino acid 15-20 of Mt MetAP1c, we aligned the amino acid sequence of the N-terminal extension region of the Mt MetAP proteins from Gram positive bacteria. Multiple sequence analysis revealed that Val-18 and Pro-19 are two highly conserved residues in different MetAPs (Figure 4A). To investigate their role, mutants replacing these two residues, one at a time (V18A, V18G, P19A, P19G) or both at the same time (V18AP19A and V18GP19G) were generated and the enzymatic activities of the recombinant proteins were monitored. Relative to wild type, V18A protein had ~30% activity while P19A and P19G exhibited ~65% and 35% activity respectively. On the other hand, V18G, V18AP19A and V18GP19G variants were devoid of any enzymatic activity (Figure 4B). Use of 10-fold excess of protein in assays, V18A, P19A and P19G mutants displayed activities almost at par (78%-112%) with the wild-type. However, use of V18G, V18AP19A and V18GP19G proteins even at increasing amounts in assays had hardly any activity (Figure 4B). These results indicated that Val-18 and/or Pro-19, the residues present in N-terminal extension (not in the catalytic domain) may contribute towards the enzymatic activity of Mt MetAP1c. Further, to monitor whether variation in the activity of the mutant proteins could be due to structural changes, far-UV CD studies were performed. The analysis of CD spectra revealed that mutations caused alteration in secondary structure of all the variants (Figure 4C). Compared to the wild type, mutation of Val-18 or Pro-19 (V18A, P19G, V18AP19A) resulted in the reduction of the negative band intensity in the region of 205-208 nm of the mutant proteins (Figure 4C). In the variant P19A, the reduction in band intensity was also observed at 222 nm (in addition to 205-208 nm), thereby indicating that the helical content was reduced. In V18G significant reduction was observed both at 205-208 nm and 222 nm. Thus, these results suggested the importance of these two residues (Val-18 and Pro-19) exclusively and mutually towards the enzymatic activity of Mt MetAP1c.

Figure 4
figure 4

Val-18 and Pro-19 at N-terminal extension are crucial for Mt MetAP1c enzyme activity. (A) Sequence alignment of MetAP1c from different gram positive bacteria. Notations used with accession number in parantheses: M tb-Mycobacterium tuberculosis (P0A5J2); M bovis-Mycobacterium bovis (P0A5J3); M avium-Mycobacterium avium (A0QJ09); M marinum-Mycobacterium marinum (B2HJQ5); M paratuberculosis-Mycobacterium paratuberculosis (Q73VS7); M leprae-Mycobacterium leprae (Q9CBU7); S coelicolor-Streptomyces coelicolor (Q9RKR2); S scabies-Streptomyces scabies (C9ZHA6); R erythropolis-Rhodococcus erythropolis (C0ZY62); N farcinica-Nocardia farcinica (Q5YSA3); G bronchialis-Gordonia bronchialis (D0LBG0); C diphtheriae-Corynebacterium diphtheriae (Q6NGL5); C glutamicum-Corynebacterium glutamicum (Q6M437); B mcbrellneri-Brevibacterium mcbrellneri (D4YNZ0); L xyli-Leifsonia xyli subsp. xyli (Q6AFH6). (B) Effect of mutations on the Mt MetAP1c enzyme activity. The indicated amounts of wild-type and mutant proteins were used to monitor the enzyme activity with 4 mM Met-Ala-Ser as the substrate. (C) Far-UV CD spectra of the different mutant proteins.

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To have an idea on how the Val-18 and Pro-19 of Mt MetAP1c influenced the activity of the enzyme, we used crystal structure of Mt MetAP1c to perform the Molecular dynamic (MD) simulation studies by introducing point mutation(s) at the site of interest (see 'Methods'). Four MD simulations were performed in explicit solvent conditions to examine the effect of specific mutations on overall structure and on the interactions of amino acids lining the active site of the protein. Comparison of the trajectories between wild-type and double mutant (V18GP19G) showed that overall fold of mutant protein is retained (Figure 5A). However, specific and drastic changes in the positions of active site residues were observed (Figures 5B and 5C). The conformational changes that initiated from the site of mutation (Val-18, Pro-19) propagated to the active site and displaced His-114 as well as affected His-212 and Glu-238. Analysis of trajectories of single mutants (V18G and P19A) illustrated that stereo chemical structural changes similar to that observed in double mutant (V18GP19G) were present in V18G (Figure 5C). On the contrary, MD simulation results of P19A appeared much similar to that of the wild-type but the presence of differential population of the ensemble at different time could possibly explain the reduction in the activity of this mutant protein (Figure 5Cright panel). Thus, the results of our MD simulation studies support our biochemical observations.

Figure 5
figure 5

Molecular dynamic simulations indicate Val-18 and Pro-19 maintain active site conformation of Mt MetAP1c. (A) Cα RMSD plot as a function of time at 300K over a period of 20 ns simulations done for wild-type and different mutant proteins. (B) MD simulations. The snapshots superimposed for wild-type (left) and V18GP19G (right) for 15 ns run. (Inset), depicts the highlighted regions of snapshots at 5ns, 7.5ns, 10ns, 15ns. (C) Snapshots of single mutant (left, V18G, blue-gray) superimposed with double mutant (pink). Both overlap each other to maximum probability compared to the wild-type protein. The loop proximal to the site of mutation is out of plane of the active site. MD simulations snapshots of single mutant (right, P19A, yellow ribbon blue sticks) superimposed with wild-type (blue-gray). Both overlap each other to maximum probability compared to the double mutant. The loop proximal to the site of mutation shows configurations similar to wild-type in 3-D space i.e. in plane to the active site.

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Discussion

MetAPs represent a unique class of metalloproteases that catalyze the co-translational removal of N-terminal methionine residue from the elongating polypeptide chain during the process of protein synthesis [2]. Since MetAP action in both prokaryotes and eukaryotes is mandatory during nascent protein synthesis, the importance of this enzyme has long been realized. Though the presence of two MetAP1s was known with the availability of the M. tuberculosis genome sequence, both of them (Mt MetAP1a and Mt MetAP1c) are active has been shown recently by others [17, 19, 20] and also in this study. However, it is still not known whether they are merely complementing each other for their functionality and therefore redundant within the genome or their presence is because of the specific needs of the bacterium. Recently, it has been reported that Mt MetAP1a knock down caused ~75% viability of mycobacteria while elimination of Mt MetAP1c resulted in 95% survival [20]. Since this enzyme from different sources has been considered as a potential drug target, identification of inhibitors against these enzymes, particularly for M. tuberculosis Mt MetAP1a is in progress [20]. However, the initial step of structure-activity relationship between these two proteins has not been addressed properly. In this context, to carry out systematic study of these two enzymes from M. tuberculosis, we have attempted here to analyze MetAP1s to elucidate subtle differences in their characteristics.

Our results indicated that Mt MetAP1c had strikingly high enzyme turnover rate with the same substrate (MAS) compared to Mt MetAP1a (~350-fold more; Table 1), although they had different substrate preferences (Figure 1A). The outcome of our CD as well as activity assays with these mycobacterial enzymes illustrate that Mt MetAP1a can sustain high temperature (up to 50°C) compared to Mt MetAP1c. In fact, Tm value calculated based on CD spectra for Mt MetAP1a was 53.6°C as opposed to 42.7°C for Mt MetAP1c (Figure 1D). To get an insight into the cause of such differential behaviour of the two enzymes, structure of Mt MetAP1a was modelled and compared with the available structure of Mt MetAP1c (Figure 2B). We observed that the active site residues in both the Mt MetAP1s are essentially same, though their arrangement in space is different (Figure 2B). Furthermore, these amino acids are crucial because any alteration in them (for both the enzymes) yielded an inactive protein (Figure 2C). Thus it is logical to presume that this difference in the arrangement may be responsible for the alteration in their activity/functionality.

Sequence analysis revealed that the remarkable feature of Mt MetAP1c compared to Mt MetAP1a, is the presence of 40 amino acid long N-terminal extension. It has been suggested that this extension may be involved in the interaction of Mt MetAP1c with the ribosome [9]. In Sc MetAP1 and Hs MetAP2 deletion of N-terminal extension did not significantly alter the catalytic efficiency of the enzymes [23–25]. However, such deletion in Sc MetAP1 reduced its efficiency in rescuing the slow growth phenotype of a map mutant [26]. Interestingly, the zinc finger motif present in the N-terminal extension of Sc MetAP1 has been shown to be involved in its association with the ribosome [26]. In Hs MetAP1, on the other hand, these sequences have been implicated for the interaction of the enzyme with substrates and inhibitors [25]. This led us to explore the role of the N-terminal extension present in Mt MetAP1c towards the activity of the enzyme. We constructed a series of deletion mutants removing nine (Δ2-10), fourteen (Δ2-15), nineteen (Δ2-20), twenty-nine (Δ2-30) and thirty-nine (Δ2-40) amino acids from the amino terminal end of the Mt MetAP1c (Figure 3A). Enzyme assays with these constructs using even 10 fold excess of proteins compared to the wild-type revealed that there was a considerable loss in activity in Δ2-10 or Δ2-15 variants and no activity at all in mutants, like Δ2-20 or Δ2-30 or Δ2-40 (Figure 3B). Intriguingly, following resolving in SDS-PAGE, analysis of the deletion constructs in Coomassie Brilliant Blue stained gels reflected that purification profile of Δ2-10 or Δ2-15 mutants was similar to that of the wild-type but variants like Δ2-20, Δ2-30 and Δ2-40 exhibited loss in purity of the proteins (Figure 3C, upper panel). Hence, it seems logical to postulate that the amino acid residues between 15 and 20 of Mt MetAP1c are crucial for its activity. Perhaps, deletion of residues beyond 19 from the N-terminal end of Mt MetAP1c renders the enzyme unstable or exposes its hydrophobic patches, thereby increasing its tendency to associate with the other proteins. In fact, evaluation of structural changes through far-UV and near-UV CD spectra revealed that up to 15 residues from the N-terminal extension of Mt MetAP1c are dispensable for the folding of the protein (Figure 3D) but required for its activity (Figure 3B). Thus, our results argue that the N-terminal extension is important for the catalytic activity of Mt MetAP1c protein, which is unlike the reports available with Sc MetAP1, Hs MetAP1 and Hs MetAP2 [23–25].

To identify the critical residue(s) for the activity of the protein, we analyzed the sequence of MetAPs from different Gram positive bacteria with N-terminal extension and found the presence of conserved Val-18 and Pro-19 in between amino acid residues 15 and 20 (Figure 4A). This observation, prompted us to create point mutations at 18th and 19th position of Mt MetAP1c (one at a time or together with type-to-type/drastic substitutions) and examine their effect on the activity of the enzyme. While V18A and P19A were partially active, V18AP19A did not show any activity. On the other hand, P19G exhibited partial activity but neither V18G nor V18GP19G were active (Figure 4B). Further, to assess if these mutations resulted in any structural alterations, far-UV CD studies were carried out. Interestingly the mutation caused alteration in the secondary structure of the protein. Thus, our results argue the importance of these two residues (Val-18 and Pro-19) exclusively and mutually towards the enzymatic activity of Mt MetAP1c. It appears very likely that the structural alterations that occurred due to mutation at Val-18 and/or Pro-19 of Mt MetAP1c affected the active site conformation of the protein. In fact, MD simulations (both 5 ns and 50 ns) indicated that the conformational changes generated at the site of mutation (V18GP19G and V18G) propagated via connecting loops and helices to the enzyme active site resulting in the alteration in the movement/positioning of residues like His-114, His-212 and Glu-238 which are critical for the enzyme activity as revealed in our biochemical studies (Figure 2C). These variations in the V18GP19G or V18G mutant proteins presumably made the active site environment unfavourable for any activity. Thus, our results illustrate that residue(s) without being in the active site of an enzyme is capable of modulating its activity, which does not seem to be unusual [27, 28].

Conclusion

Our findings unequivocally emphasize that N-terminal extension of Mt MetAP1c contributes towards the functionality of the enzyme by regulating active site residues through "action-at-a-distance" mechanism and this is presumably its unique function in Mt MetAP1c, which we are reporting for the first time. Since Val-18 and Pro-19 are conserved residues throughout the Gram positive bacterial MetAP1s with N-terminal extension, it remains to be seen whether they are universal in contributing towards the functionality of the enzyme.

Methods

Materials

Restriction/modifying enzymes were obtained from New England Biolab. All other fine chemicals including Met-Gly, Met-Ala-Ser, Met-Gly-Met-Met, Gly-Gly-Ala were procured from Sigma Chemical Company. Ni-NTA resin (Qaigen), ECL Western blotting detection kit, PCR DNA/gel band purification kit, protein molecular weight markers (GE Healthcare) and Herculase fusion DNA polymerase (Stratagene) were commercially available. Oligonucleotides used in this study were custom synthesized (IDT/Ocimum Biosolutions/Sigma).

DNA manipulation and generation of Mt MetAP-mutants

Genes encoding two MetAP1, mapA (Rv0734) and mapB (Rv2861c) of M. tuberculosis H37Rv (virulent strain) exhibited 100% identity at the nucleotide level with its avirulent strain (H37Ra). In this study, genomic DNA isolated [29] from M. tuberculosis strain H37Ra was used for PCR amplification of mapA and mapB genes. For this purpose, gene specific primers incorporating restriction sites were designed (primers CK74 with HindIII and CK140 with NheI sites for mapA; primers CK64 with NdeI and CK65 with HindIII sites for mapB; see Additional file 1, Table S1 for primer sequences) based on published genome sequence [30] and PCR reaction was carried out using Herculase fusion DNA polymerase following manufacturer's recommended protocol. This was followed by cloning of genes at NheI/HindIII (for mapA) or NdeI/HindIII (for mapB) sites in expression vector (pET-28c) using standard protocols [31] to obtain pET-mapA or pET-mapB construct. PCR was also employed for generation of different mutations (H88A, H193A, D117A, E219V and W236L for Mt MetAP1a and H114A, H212A, D131A, E238A, W255L, V18A, V18G, P19A, P19G, V18AP19A, V18GP19G for Mt MetAP1c) including deletion mutants (Δ2-10, Δ2-15, Δ2-16, Δ2-17, Δ2-18, Δ2-19, Δ2-20, Δ2-30, Δ2-40). For each point mutation two external (CK74/CK140 and CK64/CK65 for mapA and mapB respectively) and two internal primers (incorporating mutation) were designed (Additional file 1, Table S1). Two sets of primary (pET-mapA/pET-mapB as the template) and one set of secondary (mixture of primary reaction products as the template) PCR reactions were carried out for each mutation. For constructing deletion mutants, pET-mapB was used as template with two set of primers, one primer incorporating NdeI site (excluding N-terminal region to be deleted) and another was external primer (CK65). All these constructs including the wild-type (in pET-28c) were individually transformed into E. coli strains DH5α to build up DNA and in BL21 (DE3) for expression/purification of His-tagged proteins. All mutations were confirmed by sequencing using an automated DNA sequencer.

Expression and purification of recombinant proteins

Cells harboring pET-mapA or pET-mapB or different mutant constructs were grown overnight (15 h at 37°C) in LB broth (50 μg/ml kanamycin) and induced with 0.4 mM IPTG at OD600 of ~0.8. Cells were harvested after 12 h following incubation at 16°C, resuspended in lysis buffer (100 mM Tris, pH 7.5 containing 300 mM NaCl for Mt MetAP1a or 200 mM NaCl for Mt MetAP1c, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml of pepstatin and 1 μg/ml of leupeptin or inhibitor cocktail, Roche) and sonicated. The supernatant fraction was further loaded on a Ni-NTA affinity column, washed with 10 bed volumes of 100 mM Tris pH 7.5 containing 300 mM (for Mt MetAP1a) or 200 mM (for Mt MetAP1c) NaCl with 20 mM imidazole. The purified protein(s) was eluted with elution buffer comprising of 100 mM Tris, pH 7.5 containing either 300 mM NaCl/250 mM imidazole (for Mt MetAP1a) or 200 mM NaCl/150mM imidazole (for Mt MetAP1c). Following removal of imidazole by dialysis at 4°C for ~14h (dialysis buffer: 100 mM Tris, pH 7.5 with either 300 mM NaCl for Mt MetAP1a or 150mM NaCl for Mt MetAP1c; buffer changed: 4 times), protein was estimated [32] and stored at -80°C until used.

Methionine aminopeptidase activity

MetAP activity was measured by a colorimetric assay performed at 30°C in a microtiter plate by monitoring the absorbance of oxidized o-dianisidine at 440 nm [33]. Since the assay system involves the use of CoCl2, we determined its optimal amount (2 mM for Mt MetAP1a and 0.2 mM for Mt MetAP1c) to be used for the assessment of enzyme activity at our experimental conditions. We found use of 2 mM CoCl2 did not inhibit Mt MetAP1a enzyme activity and similar amount has also been used for E. coli MetAP enzyme assay [34]. Briefly, in a 100 μl reaction volume Mt MetAP1a (1.7 nM/reaction) or Mt MetAP1c (0.038 nM/reaction) protein was mixed with a reaction mixture consisting of 100 mM Tris-HCl pH 7.5, 0.2 mM (for Mt MetAP1c) or 2 mM (for Mt MetAP1a) CoCl2, 0.1mg/ml o-dianisidine, 3 units of horse radish peroxidase, 0.5 units of L-amino acid oxidase and 0.75-8 mM substrate (Met-Ala-Ser or Met-Gly-Met-Met). The activity of the enzyme was monitored in an ELISA plate reader for 2-15 min. The values obtained were corrected by subtracting the blank readings (no significant difference was noticed when assays were carried out with all ingredients except either substrate or protein) and μmol of product released was calculated by using the extinction coefficient [35] of oxidised o-dianisidine as 10580 M-1cm.-1.

Western blotting

Purified proteins (1 μg protein/slot) were resolved in 12% SDS-PAGE and transferred to nitrocellulose membranes (0.45 μm) using a mini-transblot apparatus (Bio-Rad) for Western blotting. Membranes were stained with Ponceau S to ensure transfer, processed using anti-His/anti-mouse IgG and detected through ECL detection system following manufacturer's (GE Healthcare) recommended protocol.

CD spectroscopy

CD spectra of Mt MetAP1a and Mt MetAP1c were recorded in a Jasco J-810 spectropolarimeter before and after thermal denaturation. Measurements in the far-ultraviolet region (190-250 nm) were performed on protein solutions (0.15-0.3 mg/ml in 20 mM Tris, pH 7.5 and 150 mM/300 mM NaCl) employing a cell with path length of 0.1 cm at 25°C. The protein samples were heated at a rate of 3°C/min up to 50°C and the spectra were recorded at 25°C and 50°C. For calculating Tm, Mt MetAP1a and Mt MetAP1c were heated in the thermoelectric cell of the CD spectrophotometer at the fixed rate of 1°C/min and the thermal denaturation curves were recorded at 208 nm. Measurements in near-ultraviolet region (250-350 nm) were carried out using cell with path length of 1cm at 25°C on protein solutions (0.3-0.5 mg/ml in 20 mM Tris, pH 7.5 and 150 mM/300 mM NaCl). The mean residue ellipticity [θ] was calculated using a mean residue molecular mass of 110 Da. Each spectrum reported is an average of ten scans. Blank spectra of aqueous buffer were used to correct the observed spectra.

Bioinformatic analysis

Nucleotide derived amino acid sequence of Mt MetAP1c was compared with non-redundant database using BLAST [36]. The multiple sequence alignment of the retrieved sequences was carried out using ClustalX [37] with default values for gap opening and extension penalties.

Molecular modelling

The crystal structure of Mt MetAP1c (PDB ID: 1YJ3) [9] served as the template for modeling of Mt MetAP1a using Modeller 9v7 program and model was selected on the basis of DOPE score [38–40]. The PROCHECK validated model was energy minimised using LEap module of AMBER 9 package [41, 42]. Structures of different mutants (V18G, P19G, and V18GP19G) were generated using Mt MetAP1c as the template and residue replacement editor in PYMOL. The structure of all the mutants were also energy minimized.

Molecular dynamics simulations

All MD simulations were performed with AMBER 9.0 and FF03 forcefield [43]. The protein was embedded in the TIP3P water box [44], which approximately extended 10Å in each direction from the peripheral surface of the protein to any periodic box edge with unit box dimensions of 18.774 × 18.774 × 18.774 Å. Periodic boundary conditions were applied and the entire system (solvent with protein) was energy minimized. Initial equilibration was monitored for 1 ns and the production MD simulation was then run at 300K, maintained using Langevin weak coupling algorithm [45]. The simulation trajectory was calculated using heavy atom harmonic position SHAKE [46]. Simulation was continued till 50 ns with an integration step of 2 fs and snapshots were retained every 10 ps for further analysis. MD trajectories were analyzed using the PTRAJ module and structures were visualized using VMD molecular visualization program [47].

Data analysis

Unless mentioned otherwise, reproducibility of each experiment throughout this study was checked at least three times and results were calculated as Mean ± SD.

Abbreviations

anti-his:

antibody against histidine-tag

IPTG:

isopropyl-β-D-thiogalactopyranoside

Hs MetAP Homo sapiens:

Methionine aminopeptidase

MD:

molecular dynamic simulations

Sc MetAP1 Saccharomyces cerevisiae:

Methionine aminopeptidase 1

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Acknowledgements and Funding

We are thankful to Dr. P. Guptasarma for his valuable inputs on the interpretation of CD data and to Dr. Haider H Dar for critical reading of this manuscript. We are indebted to Mr. Gaurav Sharma for his help during the course of this study. We are thankful to Mr. J. Prasad for providing us with excellent technical assistance whenever sought. Research Fellowships (P.K. and M.M.) and grants (PKC) from the DBT/CSIR, New Delhi, India are gratefully acknowledged.

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  1. Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh, 160 036, India

    Pavitra Kanudia, Monica Mittal, Sangaralingam Kumaran & Pradip K Chakraborti

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  1. Pavitra Kanudia
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  2. Monica Mittal
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Correspondence to Pradip K Chakraborti.

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PK and PKC conceived the study, planned the experiments, analyzed results and wrote the paper. PK did all wet lab experiments. MM and SK carried out Molecular modelling and Molecular dynamic simulation studies, interpreted results and wrote respective portions of the paper. All authors read and approved the final manuscript.

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Kanudia, P., Mittal, M., Kumaran, S. et al. Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity. BMC Biochem 12, 35 (2011). https://doi.org/10.1186/1471-2091-12-35

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