How metal cofactors drive dimer–dodecamer transition of the M42 aminopeptidase TmPep1050 of Thermotoga maritima

Abstract

The M42 aminopeptidases are dinuclear aminopeptidases displaying a peculiar tetrahedron-shaped structure with 12 subunits. Their quaternary structure results from the self-assembly of six dimers controlled by their divalent metal ion cofactors. The oligomeric-state transition remains debated despite the structural characterization of several archaeal M42 aminopeptidases. The main bottleneck is the lack of dimer structures, hindering the understanding of structural changes occurring during the oligomerization process. We present the first dimer structure of an M42 aminopeptidase, TmPep1050 of Thermotoga maritima, along with the dodecamer structure. The comparison of both structures has allowed us to describe how the metal ion cofactors modulate the active-site fold and, subsequently, affect the interaction interface between dimers. A mutational study shows that the M1 site strictly controls dodecamer formation. The dodecamer structure of TmPep1050 also reveals that a part of the dimerization domain delimits the catalytic pocket and could participate in substrate binding.

Introduction

Cells possess an arsenal of proteolytic enzymes to ensure specific and nonspecific hydrolysis of proteins and peptides (for instance, up to 2% of the human genome encodes proteases) (1). Proteolysis plays an important role in a wide array of cellular functions as diverse as homeostasis, stress responses, cell-cycle regulation, quorum sensing, stalled ribosome rescue, immune response, virulence, etc. (2–6). About 80% of protein degradation is achieved by the proteasome 26S in eukaryotes (3). Protein turnover is carried out by either the proteasome 20S in archaea and actinomycetes or several proteolytic complexes, notably HslUV, ClpAP, ClpXP, Lon, and FtsH, in other prokaryotes (4, 7). The proteasome and its related complexes generate peptides 6–9 amino acids in length (8) that are further processed by a set of endopeptidases and exopeptidases. Several peptidases have been proposed to act downstream of the proteasome: the tricorn protease (9), thimet oligopeptidase (10), tripeptidyl peptidase II (11), aminopeptidase N (12), and TET² aminopeptidases (13). The latter peptidases are widely distributed in all kingdoms of life (14). They adopt a peculiar tetrahedron-shaped structure compartmenting the active sites in a buried catalytic chamber (15). In Pyrococcus horikoshii, four TET aminopeptidases, PhTET1, PhTET2, PhTET3, and PhTET4, have been described, and each of them displays a different substrate specificity, aspartyl, leucyl, lysyl, and glycyl aminopeptidase activity, respectively (15–18). Remarkably, heterocomplexes, made of PhTET2 and PhTET3, have been reported, leading to the assumption of the existence of a peptidasome particle (19, 20).

According to MEROPS classification, the TET aminopeptidases are found in the M18 and M42 families (14, 21). Both families belong to the MH clan, encompassing metallopeptidases sharing a common α/β catalytic domain. The archetypal MH clan enzyme is the Vibrio proteolyticus aminopeptidase 1 (22). The M18 family is widely distributed in all domains of life (14), whereas the M42 family is unique to prokaryotes (23). Several structures of archaeal M42 aminopeptidases have been studied (13, 15, 17, 18, 24–26), but only one structure has been reported for bacteria (27). Franzetti et al. (25) described the first TET aminopeptidase structure of the Haloarcula marismortui M42 aminopeptidase, consisting of 12 subunits adopting a tetrahedron-shaped quaternary structure. High-resolution structures of P. horikoshii PhTET2 have revealed that the M42 aminopeptidase subunit is composed of an α/β catalytic domain and a PDZ-like dimerization domain (13, 15). A similar quaternary structure has been reported for several M18 aminopeptidases from prokaryotes and eukaryotes (28–31), although their dimerization domain adopts a butterfly fold instead of a PDZ-like fold. The quaternary structure is often seen as the assembly of six dimers with a dimer positioned on each edge of the tetrahedron (13). At the center of the tetrahedron faces are four gates leading to a wide inner cavity. Four exit pores are located at the tetrahedron vertices through which amino acids, generated during peptide hydrolysis, are released.

The catalytic site is characterized by (i) two catalytic residues, (ii) five residues forming the metal ion–binding sites M1 and M2, and (iii) two divalent metal ions (usually Zn²⁺, Co²⁺, and Mg²⁺). The metal ions have been described to bind the substrate, to facilitate nucleophile generation (water molecule deprotonation), and to stabilize the transition state of peptide hydrolysis (32). In addition to their catalytic roles, the metal ions could also control the TET aminopeptidase oligomerization. Such a structural role has been reported for three M42 aminopeptidases, PhTET2 and PhTET3 from P. horikoshii (13, 33, 34), and PfTET3 from Pyrococcus furiosus (24). Under chelating conditions, dodecamers disassemble into either dimers (PhTET3) or monomers (PhTET2 and PfTET3). Colombo et al. (24) inferred the role of the M1 and M2 sites in the oligomerization: the presence of a metal ion in the M2 site is required for the oligomerization, whereas the M1 site controls protein flexibility. According to Macek et al. (33), the dissociation is reversible as dodecamers are formed through random association of dimers. The understanding of the dodecamer formation mechanism, however, suffers from the lack of dimer (or monomer) structure. A low-resolution structure of PhTET2 dimer has been reported, but no significant structural change has been pinpointed (35). The current knowledge on M42 aminopeptidases relies on a set of archaeal enzymes, whereas their bacterial counterparts remain ill-described.

In this work, we focus on the bacterial M42 aminopeptidase model, TmPep1050 from Thermotoga maritima, previously characterized as a cobalt-activated leucyl aminopeptidase (23). We present the first high-resolution dimer structure of an M42 aminopeptidase alongside the dodecamer structure. The dimer structure clearly showed the cofactor role in the active-site fold that reverberates the interaction interfaces between dimers. In addition, the thermostability of TmPep1050 depends on the oligomeric state. The dodecamer assembly/disassembly was further studied by native MS, revealing the intermediate oligomeric states as well as the flexible parts in the dimer structure. Finally, we inferred the role of the M1 and M2 sites through the structural study of TmPep1050 variants. Our results showed that the M1 site strictly controls the dodecamer assembly. Based on the current knowledge, we should avoid drawing general dogma on the TET aminopeptidases due to the lack of studies on metal ion binding behavior and structures in complex with substrates and inhibitors.

Results and discussion

TmPep1050 has a genuine TET aminopeptidase structure

The Tm_1050 open reading frame (ORF) was overexpressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity through three chromatographic steps. Size-exclusion chromatography showed that the purified enzyme, subsequently named TmPep1050_12-mer, had an apparent molecular weight of 330 ± 15 kDa (S.E. with n = 12; molecular weight of a monomer, 36.0 kDa). Native MS analysis demonstrated that TmPep1050_12-mer is a dodecamer with a measured mass of 435,323 ± 64 Da (theorical mass, 432,783 Da). Crystals were obtained in two crystallization conditions (see Table S1). X-ray fluorescence scanning detected Co²⁺ and Zn²⁺ in crystals grown in 0.18 m triammonium citrate, 40% PEG3350, pH 7.5, whereas no trace of metal ions was found in crystals grown in 2.1 m malic acid, pH 6.75 (see Fig. S1). The absence of metal ions could be due to the use of malic acid, a weak chelating agent of divalent metal ions (K_d, 10⁻³ m for Co²⁺ binding) (36), in the crystallization buffer. Under such conditions, TmPep1050_12-mer was barely active, although dodecameric oligomerization remained unaffected at room temperature (see below). A similar statement has been reported for PhTET2 (13): when cocrystallized with o-phenanthroline, PhTET2 remained dodecameric even if the two Zn²⁺ were chelated. The apoenzyme structure (apo-TmPep1050_12-mer) was determined by molecular replacement using the coordinates of YpdE, an uncharacterized M42 aminopeptidase of Shigella flexneri. The metal-bound complex structure was determined by MR and single-wavelength anomalous diffraction using apo-TmPep1050_12-mer coordinates.

The TmPep1050_12-mer quaternary structure consists of 12 subunits adopting a tetrahedron-shaped architecture (see Fig. 1A) like other available structures of M42 aminopeptidases (13, 17, 18, 24, 26, 27). The tetrahedron-shaped architecture is often described as the self-assembly of six dimers in such a manner that a dimer lies on each tetrahedron edge. Four entrances are located at the center of the faces, and four exit channels are located at the vertices. The entrances and exits lead to an inner cavity with the 12 catalytic sites oriented inward, compartmenting the active sites. The interaction between dimers is maintained through a polar and hydrophobic interaction network at the vertices (see Fig. S2A) and nine salt bridges between adjacent subunits (see Fig. S2B). The residues forming these salt bridges are, however, not conserved among all M42 aminopeptidases. The apo-TmPep1050_12-mer structure appears to be nearly identical to the TmPep1050_12-mer structure obtained with bound metal ions (structural alignment r.m.s.d., 0.235Å). There are, however, several differences observed in the catalytic site, and they are described below.

Figure 1.

The TmPep1050_12-mer structure. A, schematic representation of TmPep1050_12-mer quaternary structure (PDB code 6NW5) centered on one of the tetrahedron faces. As the asymmetric unit contains four monomers, the quaternary structure was reconstituted by PDBePisa. B, schematic representation of TmPep1050_12-mer subunit. The α4 helix, β16 strand, and β sheet extension (β10, β11, and β17) are colored in green, dark blue, and purple, respectively. Zn²⁺ and Co²⁺ are displayed as gray and pink spheres, respectively. C, schematic representation of subunits A and B (red and blue, respectively) composing a dimer in the TmPep1050_12-mer structure. The subunit A catalytic pocket is indicated by an arrow, the α4 helices are schematized as cylinders. D, structural alignment of the five metal ion–binding residues and the two catalytic residues of TmPep1050_12-mer (in light gray) and apo-TmPep1050_12-mer (PDB code 4P6Y; in light blue). Zn²⁺, Co²⁺, and H₂O are represented as gray, pink, and red spheres, respectively. The distance between His-60 and His-307 Nϵ2 is about 6.7 Å in the TmPep1050_12-mer structure. In the apo-TmPep1050_12-mer structure, this distance is increased to about 8.2 Å.

The α4 helix of the PDZ-like domain delimits the TmPep1050_12-mer catalytic pocket

The TmPep1050_12-mer subunit is composed of a catalytic domain and a dimerization domain (Fig. 1B). The catalytic domain adopts an α/β globular structure similar to that of V. proteolyticus aminopeptidase Ap1 (see Fig. S3) except that the β sheet displays an extension of three antiparallel strands (β10, β11, and β17) connected to the β16 strand. The β sheet extension is conserved among the M42 family (13, 15, 21) and interacts with the neighbor subunit dimerization domain (13, 27). The TmPep1050_12-mer dimerization domain adopts the typical fold of a PDZ-like domain (13). In all structurally characterized M42 aminopeptidases, the α4 helix of the PDZ-like domain is highly flexible (13, 15, 17, 18, 24, 26, 27). The α4 helix, however, was modeled in TmPep1050_12-mer structure, probably due to its stabilization in the crystal. In the quaternary structure, the α4 helices are positioned in such a manner that the entrances are restricted to 13 Å (see Fig. S4), which is smaller than those reported for PhTET1, PhTET2, and SpPepA (13, 18, 27). The carboxylate functions of three glutamate residues (Glu-110, Glu-114, and Glu-117) are oriented toward the entrance. In addition, the TmPep1050_12-mer active sites appear to be more buried and delimited than those of other characterized M42 aminopeptidases as the α4 helix of one monomer delimits the active-site pocket of the other monomer at the dimer level (see Figs. 1C and S5A). Hence, the α4 helix flexibility could indicate a possible open/closed conformation of the enzyme. It is worth noticing that an α helix of the butterfly-fold dimerization domain of the M18 aminopeptidases could also be highly flexible (30, 31) or, to the contrary, more stable (28, 29). Such an α helix, flexible or not, seems to be a conserved structural feature among TET aminopeptidases. In the human M18 aminopeptidase structure, this α helix is directly involved in substrate binding (28). A similar function could be expected for the α4 helix in M42 aminopeptidases.

Seven residues conserved in the MH clan define the TmPep1050_12-mer active site

The M42 aminopeptidase catalytic site consists of two divalent metal ions (M1 and M2) bound by five strictly conserved residues and two catalytic residues. In the TmPep1050_12-mer catalytic site, the M1 site consists of Asp-168, Glu-198, and His-307 residues bound to a Co²⁺ ion, whereas the M2 site consists of His-60, Asp-168, and Asp-220 residues bound to a Zn²⁺ ion (see Fig. 1D). In the apo-TmPep1050_12-mer structure, these residues are correctly positioned despite the absence of metal ions. The distance between the imidazole rings of His-60 and His-307 is, however, increased by 1.5 Å, whereas the carboxylate of Glu-198 is displaced by 0.4 Å away from the M1 site center compared with the TmPep1050_12-mer structure with its metallic cofactors. Thus, the presence of metal ions seems to pull the side chains of metal-binding residues closer, especially those of the M1 site (see Fig. 1D). The two metal ions also coordinate a water molecule involved in peptide bond hydrolysis. The water molecule is asymmetrically positioned, being closer to the M2 site rather than the M1 site (see Fig. S5B). In the monomer D, the distance between Zn²⁺ and the oxygen atom is so short that only a hydroxide ion could be modeled instead of a water molecule, suggesting that the M2 site stabilizes the hydroxide ion prior to the nucleophilic attack. The TmPep1050_12-mer structure contrasts with V. proteolyticus Ap1, PhTET1, and PhTET2 where the water molecule is positioned symmetrically (see Fig. S5B). In the TmPep1050_12-mer catalytic site, the acid/base catalyst is Glu-197, which is conserved in all MH clan members. Indeed, the mutation of Glu-197 to glutamine completely abolished the activity (specific activity of l-Leu-pNA of less than 0.1 s⁻¹), whereas the oligomeric state remained unaffected. The seventh conserved residue is Asp-62, which has been described as a modulator of Lewis acid strength of M2 in V. proteolyticus Ap1 (37). The TmPep1050_12-mer S1-binding pocket is similar to that of PhTET2 because six of seven residues are conserved (see Fig. S5C), which is in accordance with their substrate specificities as both enzymes are leucyl aminopeptidases.

TmPep1050_12-mer is a cobalt-activated leucyl aminopeptidase

TmPep1050_12-mer aminopeptidase activity was assayed with various l-aminoacyl-p-nitroanilide (pNA) derivatives. The substrate specificity is mainly toward nonpolar aliphatic l-aminoacyl-pNA, with a clear preference for l-Leu-pNA (Table 1). TmPep1050_12-mer activity is maximal at pH between 7.0 and 7.8 and up to 90 °C (see Figs. S6 and S7). Its kinetic parameters, k_cat and K_m, were determined for l-Leu-pNA, l-Ile-pNA, and l-Met-pNA (Table 2). These values differ greatly from our previous results obtained with His-tagged TmPep1050 (23), indicating that the polyhistidine tag could interfere in the binding of divalent metal ions to the M1 and M2 sites. Indeed, we reported a k_cat of 0.25 s⁻¹ for His-tagged TmPep1050, even with a Co²⁺-to-enzyme ratio of 500.

Table 1.

TmPep1050_12-mer aminopeptidase activity against l-aminoacyl-pNA derivatives

k, specific activity; ND, not detectable (k < 0.05 s⁻¹).

Substrates	k
	s−1
l-Leu-pNA	118.3 ± 3.6
l-Ile-pNA	42.9 ± 1.6
l-Val-pNA	7.2 ± 0.8
l-Met-pNA	6.3 ± 0.2
l-Phe-pNA	0.9 ± 0.02
l-Ala-pNA	0.1 ± 0.01
l-Glu-pNA	ND
Gly-pNA	ND
l-His-pNA	ND
l-Lys-pNA	ND
l-Pro-pNA	ND
Ac-Leu-pNA	ND

Table 2.

TmPep1050_12-mer kinetic parameters

The ratio k_cat/K_m represents the catalytic efficiency.

Substrates	K	kcat	kcat/K
	μm	s−1	s−1 m−1
l-Leu-pNA	1750 ± 250	138 ± 12	7.9 × 104
l-Ile-pNA	1100 ± 100	53 ± 4	4.8 × 104
l-Met-pNA	1750 ± 100	16 ± 1	9.1 × 103

Dialysis of TmPep1050_12-mer against 2.1 m malic acid, pH 7.0 (one of the crystallization conditions), resulted in a loss of activity (dropping to 1 s⁻¹), whereas its oligomeric state remained unaffected as determined by gel filtration with an apparent molecular weight of 300 ± 10 kDa (S.E. with n = 3). The addition of Co²⁺ partly restored TmPep1050_12-mer LAP activity, whereas Zn²⁺ had no effect on LAP activity, although it was found in the active site (see Table 3). Similar observations have been reported for other M42 aminopeptidases: although their structures contain Zn²⁺, PhTET2, PhTET3, PfTET3, and SpPepA are activated by Co²⁺ (13, 17, 24, 27, 38). Other metal ions had no significant effect on TmPep1050_12-mer LAP activity (see Table 3).

Table 3.

Effect of divalent metal ions on TmPep1050_12-mer LAP aminopeptidase activity

Metal ion–depleted TmPep1050_12-mer was diluted at 20 μm in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2, supplemented with 1.28 mm of various metal chlorides. After 24-h incubation at 75 °C, specific activities (k) were measured with l-Leu-pNA as substrate.

Metal ion	k
	s−1
Co2+	40.1 ± 1.1
Zn2+	1.9 ± 0.02
Mn2+	1.3 ± 0.02
Mg2+	1.4 ± 0.01
Ni2+	1.1 ± 0.03
Cu2+	1.4 ± 0.01
Ca2+	1.6 ± 0.01

To quantify its affinity for Co²⁺, metal ion–depleted TmPep1050_12-mer was incubated with an increasing amount of Co²⁺, ranging from 0 to 2560 μm. After 24-h incubation at 75 °C, Co²⁺ binding was quantified with an Amplex UltraRed fluorescent probe. In parallel, LAP activity was measured to follow the reactivation of metal-depleted enzyme (see Fig. 2). Co²⁺ content of metal ion–depleted enzyme was less than one atom per 10 monomers (1.5 μm Co²⁺ for 20 μm proteins). TmPep1050_12-mer can bind Co²⁺ with an apparent association constant of 50 ± 5 μm.

Figure 2.

Evolution of bound Co²⁺ per molecule of enzyme (mol. enz.) (open circles) and LAP activity (k; closed circles) in response to an increasing Co²⁺ concentration ([Co²⁺]) ranging from 0 to 2560 μm. Error bars represent S.E. with n = 3.

The thermostability and oligomerization state of TmPep1050 are Co²⁺-dependent

As TmPep1050_12-mer displays a LAP activity up to 90 °C, we expected TmPep1050_12-mer to be a highly thermostable enzyme. Its thermostability was determined at 75 and 95 °C: at 95 °C the half-life of TmPep1050_12-mer is about 24 h, whereas at 75 °C it is about 20 days. TmPep1050_12-mer is highly thermostable, probably due to many ionic and hydrophobic interactions between dimers maintaining the whole quaternary structure in dodecamers. Shorter half-lives (a few hours) have been reported for various M42 aminopeptidases (17, 38–41).

Remarkably, in the absence of its metal ion cofactors, the half-life at 75 °C is dramatically reduced to merely 1 h. Moreover, under these conditions, TmPep1050 dodecamers dissociated to dimers (namely TmPep1050_2-mer) after 2-h incubation at 75 °C as an apparent molecular weight of 52 ± 3 kDa (S.E., n = 12) was measured by gel filtration. Thermal shift assays were conducted to further characterize the role of cobalt ions in TmPep1050_12-mer thermostability. Cobalt-loaded TmPep1050_12-mer had a T_m of about 97 °C, confirming its remarkable thermostability. After dialysis against 2.1 m malic acid, pH 7.0 (as mentioned above), the T_m dropped to 91 °C; meanwhile Co²⁺ addition restored its thermostability (see Fig. 3).

Figure 3.

Thermal shift assays of Co²⁺ effect on TmPep1050_12-mer thermostability. Metal ion–depleted TmPep1050_12-mer was diluted to 20 μm in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2, supplemented with [Co²⁺] ranging from 0 to 2560 μm. After 24-h incubation at 75 °C, the thermostability was determined by measuring T_m. Error bars represent S.E. with n = 3.

The link between the metal cofactors and thermostability has been reported for several M42 aminopeptidases: Geobacillus stearothermophilus aminopeptidase I (42), Thermococcus onnurineus deblocking aminopeptidase (43), and PhTET3 (34). The dodecamer dissociation has often been achieved in harsh conditions such as EDTA treatment (24, 34, 42) or in acidic buffer (pH < 4) (33). Dimer formation has been reported for PhTET3 (34) and PhTET2 (13), whereas a further breakdown of dimers into monomers has been achieved for PfTET3 (24) and PhTET2 (33). Neither of these studies have, however, described the dodecamer dissociation in physiologically compatible conditions. Although our results focused on TmPep1050_12-mer treated with malic acid, the same dissociation of dodecamers into dimers was achieved with an extensive dialysis against 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2, followed by a heat treatment at 75 °C (see Table S2). Therefore, our data tend to support a dimer–dodecamer equilibrium depending on metal cofactor availability.

The oligomeric-state transition is reversible and Co²⁺-driven

Thermal shift assays suggested that dodecamer dissociation could be reversible, but the different oligomeric states occurring during the association/dissociation process had to be identified. For that purpose, we opted for native MS. After a buffer exchange in ammonium acetate, TmPep1050_12-mer retained its oligomeric state (molecular weight, 300 kDa according to gel filtration) but had a LAP activity reduced by two-thirds, suggesting that metal cofactors were partly lost. Native MS showed that, under these conditions, TmPep1050 was mainly a mixture of dimers and dodecamers (see Fig. 4A). MS/MS analysis of dodecamer +46 peak revealed two masses for dissociated monomer of 36,076.2 ± 1.7 and 36,136.9 ± 4.4 Da (see Fig. S8). The first mass is almost equal to the theoretical molecular weight of 36,065 Da, whereas the second mass could correspond to a monomer with one cobalt ion bound.

Figure 4.

Oligomeric state determination by native MS. A, mass spectrum of TmPep1050_12-mer at a concentration of 50 μm (trap collision energy set to 10 V). B, mass spectra of TmPep1050_12-mer in the presence of an increasing Co²⁺ concentration. 20 μm enzyme was incubated for 30 min at 75 °C with Co²⁺ at a concentration ranging from 0 to 120 μm. C, ion-mobility mass spectra of TmPep1050_12-mer (50 μm). D, mass spectra of TmPep1050_2-mer (upper) and its reassociation into dodecamers (lower). For the reassociation experiment, 20 μm enzyme was incubated with 120 μm Co²⁺ for 30 min at 75 °C.

When samples were loaded with Co²⁺ and heated at 75 °C for 30 min prior to MS analysis, peaks corresponding to dimers tended to disappear in favor of dodecamer peaks (see Fig. 4B). MS analysis of Co²⁺-reloaded TmPep1050_12-mer showed that monomer had a mass about 176 Da higher than the theoretical molecular weight. Such difference could be explained by the presence of at least two cobalt ions per subunit. In addition, MS experiments showed the existence of intermediate oligomeric states: tetramers, hexamers, and octamers (see Fig. 4C). The self-assembly pathway of M42 aminopeptidases is still poorly understood. Appolaire et al. (35) proposed that dimers self-assemble into dodecamers via intermediate hexamers for PhTET2. Macek et al. (33) debated this theory arguing that dimers self-assemble randomly to form tetra-, hexa-, octa-, and decamer intermediates for PhTET2. Our native MS data suggest that a dodecamer could result from the association of either two hexamers or a tetramer and an octamer. Such a transition of oligomeric states could occur and be controlled in vivo due to the low availability of divalent metal ions.

Native MS analysis of TmPep1050_2-mer (Co²⁺-depleted and heat-treated) revealed peaks corresponding to dimers only (see Fig. 4D). The addition of Co²⁺ combined with a heat treatment at 75 °C completely changed the mass spectra. Indeed, dimer peaks became almost undetectable, whereas dodecamers were the most abundant oligomer in the presence of a 6× excess of cobalt (see Fig. 4D). In comparison, only partial reassociation was reported for PhTET3 (34) and PhTET2 (35). A second experiment was conducted to quantify the different oligomers occurring during the reassociation. TmPep1050_2-mer was incubated with an increasing concentration of Co²⁺ at 75 °C for 30 min, and the oligomers were identified by size-exclusion chromatography (see Fig. 5A). The intermediate oligomers were not detected due to their low abundance (as seen in MS experiments). As expected, the ratio of dodecamers to dimers increased according to the Co²⁺ concentration (see Fig. 5, A and B).

Figure 5.

Reassociation of dimers into dodecamers in response to Co²⁺ concentration. TmPep1050_2-mer was incubated with Co²⁺ at a concentration ranging from 0 to 5 mm for 30 min at 75 °C. Oligomers were detected and quantified by gel filtration on a Superdex 200 column (volume of 120 ml). A, gel-filtration chromatogram of TmPep1050_2-mer after incubation with increasing Co²⁺ concentrations of 0 (black dashes), 0.05 (light gray dashes), 0.1 (gray dashes), 0.2 (light gray dots), 0.5 (gray dots), 1 (light gray line), and 5 mm (gray line). The peak at elution volume (V_e) of 95.0 ml corresponds to dimers, whereas the peak at V_e of 81.8 ml corresponds to dodecamers. Inset, the calibration of the Superdex 200 column with albumin (Ab), conalbumin (C), aldolase (Ad), ferritin (F), and thyroglobulin (T) as standards. The correlation between V_e (ml) and the logarithm of the relative mass (M_r) is linear with R² of 0.91. The 95% confidence intervals of the linear regression are shown in dots. B, ratio of dodecamers to dimers (R_12/2) after incubating TmPep1050_2-mer with Co²⁺. Ratios were calculated based on the peak areas. Abs, absorbance. mUA, milli unit of absorbance. Error bars represent S.E. with n = 3.

TmPep1050_2-mer structure highlights the structural changes triggered by metal ion loss

We showed that cobalt ions are important for activity, thermostability, and oligomerization of TmPep1050. Native MS experiments demonstrated the reversibility of dodecamer dissociation and how it depends on metal cofactors. As dimers are unable to form dodecamers in the absence of cobalt, structural changes must occur at oligomerization interfaces. To understand these changes, the structure of TmPep1050_2-mer was solved by X-ray crystallography.

The overall shape of TmPep1050_2-mer does not differ from a dimer in the TmPep1050_12-mer structure (see Fig. 6A). However, several structural dissimilarities having a dramatic impact on the oligomerization capability are observed. Two segments are too flexible to be modeled in the TmPep1050_2-mer structure: Gly-203–Gly-208 and Phe-279–Glu-292 corresponding to the α8 and α10 helices, respectively. To rule out a potential crystallographic artifact linked to protein stacking in the crystal, TmPep1050_2-mer backbone flexibility was probed by collision-induced dissociation. Mass spectra showed that proteins were fragmented at two preferential regions, Gly-203–Pro-212 and Asn-283–Thr-290, indicating the soundness of the crystallographic data (see Fig. 6B). At each vertex of the tetrahedron-shaped dodecamer, the α8 and α10 helices of three adjacent subunits form the exit tunnel via an interaction network between Tyr-209 and Arg-289 (see Fig. S2A). This interaction network has been described to be highly important for oligomerization (21, 35). In the TmPep1050_2-mer structure, interaction between Tyr-209 and Arg-289 is prevented as Tyr-209 is completely buried.

Figure 6.

The TmPep1050_2-mer structure. A, structural alignment of TmPep1050_2-mer subunit (PDB code 5NE6; blue) versus TmPep1050_12-mer subunit (PDB code 6NW5; light gray). Arrows point to the structural dissimilarities. The metal-binding residues, catalytic residues, and residues involved in the oligomerization are shown in stick representation. B, MS fragmentation pattern of TmPep1050_2-mer under 125-V collision trap energy. In the inset, preferential fragmentations are annotated in blue. The sequences underlined are the unmodeled flexible parts of the TmPep1050_2-mer structure. C, close-up of the TmPep1050_2-mer (blue) active site versus the TmPep1050_12-mer active site (light gray). The side chains of the metal-binding residues and the catalytic residues are represented as sticks. The metal cofactors of TmPep1050_12-mer, Co²⁺ and Zn²⁺, are shown as pink and gray spheres, respectively.

Structural alignment between dimer subunit and dodecamer subunit shows that the Gln-196–Val-202, Lys-229–Ala-235, and Lys-247–Ser-254 segments diverge greatly (see Fig. 6A). High B-factors were observed for these segments, indicating that they are highly flexible (see Fig. S9). The Gln-196–Val-202 segment contains two conserved residues of the catalytic site, Glu-197 and Glu-198, which are the catalytic general base and a metal ion–binding residue of the M1 site, respectively. In the TmPep1050_2-mer structure, the Glu-197–Gly-200 loop is so disordered that Glu-197 and Glu-198 side chains point outward from the catalytic site, which explains why the dimers are far less active than the dodecamers (see Fig. 6C). Thus, metal ion binding in the M1 site is strongly impaired in TmPep1050_2-mer. The displacement of Glu-197 and Glu-198 probably has an impact on His-60 and Asp-62 as their predicted pK_a values increase from 8.5 to 10.1 and from 2.6 to 5.0, respectively. His-60 plays a pivotal role in an H-bond network in the dodecamer subunit, interacting with Asp-62, Asp-168, Asp-169, Glu-197, Glu-198, and Asp-220. In the TmPep1050_2-mer structure, the whole H-bond network is disrupted. In the dodecamer structure, the Gln-196–Val-202 loop probably imposes the fold of the subsequent α8 helix via an entangled network of H-bonds and polar interactions. In addition, the Gln-196–Val-202 segment is closely connected to the α10 helix.

The Lys-229–Ala-235 and Lys-247–Ser-254 segments are important for oligomerization as Lys-232, Arg-233, and Arg-249 are involved in the formation of salt bridges in the dodecamer (see Fig. S1B). Systematic mutagenesis of these residues was set up to support the role of salt bridges in the dodecamer structure. Single point mutations of Lys-232, Arg-233, and Arg-249 to alanine residues did not impact the oligomerization (data not shown). However, the triple mutation to either alanine or glutamate residues (TmPep1050_{K232A/R233A/R249A} and TmPep1050_{K232E/R233E/R249E}, respectively) greatly disturbed the formation of dodecamers. Indeed, the oligomeric state of TmPep1050_{K232A/R233A/R249A} is mainly dimeric as shown by gel filtration (see Fig. 7). It also forms dodecamers and tetramers representing about 32 and 20% of the purified sample, respectively. For TmPep1050_{K232E/R233E/R249E}, the mutations have a dramatic impact on the oligomerization as the dimeric fraction represents about 88% of the purified sample (see Fig. 7). TmPep1050_{K232A/R233A/R249A} has a reduced specific activity on l-Leu-pNA of 37.2 ± 0.9 s⁻¹, whereas TmPep1050_{K232E/R233E/R249E} is barely active with a specific activity of 0.15 ± 0.01 s⁻¹. Our results strongly support the role of Lys-232, Arg-233, and Arg-249 to stabilize the dodecameric structure. Nevertheless, one should avoid generalizing such a conclusion to the whole M42 family as these residues are not conserved. Appolaire et al. (35) reported the destabilization of PhTET2 by mutating five residues involved in interdimer interactions. However, in their study, the destabilization was only transitory as PhTET2 dimers slowly reassembled into dodecamers over time (35).

Figure 7.

Oligomeric states of TmPep1050_{K232A/R233A/R249A} (plain line) and TmPep1050_{K232E/R233E/R249E} (dots) determined by gel filtration on Superose 6 column. The peaks at V_e of 16.2, 17.1, and 18.3 ml correspond to dodecamers (347 kDa), tetramers (151 kDa), and dimers (54 kDa), respectively. Inset, the calibration of the Superose 6 column with albumin (Ab), conalbumin (C), aldolase (Ad), and ferritin (F) as standards. The correlation between V_e (ml) and the logarithm of the relative mass (M_r) is linear with R² of 0.99. The 95% confidence intervals of the linear regression are shown in dots. Abs, absorbance. mUA, milli unit of absorbance.

The metal ion cofactors are directly involved in structural changes during dodecamer dissociation

The TmPep1050_2-mer structure shows how the loss of metal ions induces dodecamer dissociation. The structural role of either M1 or M2 sites, however, was not so obvious as Glu-198 (M1 site) interacts with His-60 (M2 site). Therefore, systematic mutagenesis of His-60 and His-307 (M1 site) to alanine residues was undertaken; the resulting variants were named TmPep1050_H60A and TmPep1050_H307A, respectively. The variants were barely active using l-Leu-pNA as substrate (specific activity of less than 0.1 s⁻¹). Their molecular states were determined by native MS: TmPep1050_H60A was a mixture of dimers and dodecamers, whereas TmPep1050_H307A formed only dimers (see Fig. 8A). Size-exclusion chromatography of TmPep1050_H60A showed that the dodecamer-to-dimer ratio was about 0.75.

Figure 8.

A, native mass spectrum of TmPep1050_H60A in 100 mm ammonium acetate. Dimers and dodecamers were the two major species observed. B, native mass spectrum of TmPep1050_H307A in 100 mm ammonium acetate. Dimers were the main oligomeric form observed. C, structural alignment of TmPep1050_2-mer (blue), TmPep1050_H60A structure (gold), and TmPep1050_H307A (green). r.m.s.d. values are 0.122 and 0.468 for TmPep1050_H60A and TmPep1050_H307A, respectively.

As TmPep1050_H307A only exists as a dimer, the M1 site is probably the most important feature leading to TmPep1050_12-mer formation. The structures of TmPep1050_H60A (dimer only) and TmPep1050_H307A were solved by X-ray crystallography. The H307A mutation provokes the same structural dissimilarities as observed in TmPep1050_2-mer (see Figs. 8B and S9). The α8 and α10 helices could not be modeled in the TmPep1050_H307A structure, whereas the α9 helix is elongated by three residues. Glu-197 and Glu-198 residues are also oriented outward from the catalytic site. The M2 site presents the same spatial arrangement as in the dimer, indicating that only the apo form was crystallized. Our data strongly support the structural role of the metal cofactors in addition to their catalytic role. Colombo et al. (24) drew the same conclusion with PfTET3 and hypothesized that the metal ion bound to the M1 site could stabilize the oligomerization.

The presence of Co²⁺ in the M2 site is not strictly required for oligomerization as TmPep1050_H60A is able to form a dodecamer. Still, the H60A mutation impeded cobalt binding in the M1 site, probably due to H-bond network disruption. The TmPep1050_H60A dimer structure afforded a better understanding of conformational changes in the Gln-196–Tyr-209 region. Glu-197–Ile-201 forms a wide loop instead of a tight U-turn. The α8 helix is completely unwound in the TmPep1050_H60A dimer (see Fig. 8B).

Mutagenesis of His-60 and His-307, which are involved in metal ion binding in the M2 and M1 sites, respectively, led to an interesting observation. In M42 aminopeptidases, the M1 site has been described to have a lower affinity to metal ion than the M2 site (15, 24). According to Colombo et al. (24), the removal of Co²⁺ from the M1 site led to a partial dissociation of PfTET3 dodecamer into monomer. The dodecamers were fully dissociated when the second metal ion in the M2 site was removed. Surprisingly, the H307A mutation abolished the dodecamer formation for TmPep1050_H307A, whereas a partial dissociation was observed for TmPep1050_H60A. Our data strongly suggest that the M1 site strictly controls the TmPep1050 oligomerization. Further studies would be required to understand how M1 and M2 sites finely tune the oligomerization according to their affinity and selectivity.

Experimental procedures

Cloning and mutagenesis of Tm_1050

The Tm_1050 ORF was amplified from BspHI-digested TmCD00089984 plasmid (Joint Center for Structural Genomics) using Pfu DNA polymerase (Thermo Fisher Scientific) and primers ocej419 and ocej420 (see Table S3). The PCR product was inserted into the pBAD vector (Thermo Fisher Scientific) by homologous recombination in E. coli according to the seamless ligation cloning extract (SLiCE) protocol (44), giving rise to pCEC43. Briefly, the insertion of the PCR product is allowed via two 30-bp sequences, homologous to the insertion site of the pBAD vector, flanking the gene of interest. The homologous recombination is mediated by using a cell extract of PPY strain, expressing the λ prophage Red recombination system. Site-directed mutagenesis was carried out following the single-primer reactions in parallel (SPRINP) protocol (45) except for the two vectors used for TmPep1050_{K232A/R233A/R249A} and TmPep1050_{K232E/R233E/R249E} production. In that case, two synthetic genes harboring the desired mutations (GeneArt, Thermo Fisher Scientific) were introduced into the pBAD vector by homologous recombination. The primers used to generate TmPep1050 variants are listed in Table S1. All genetic constructs were verified by sequencing (Genetic Service Facility, University of Antwerp) and are listed in Table S4. The E. coli MC1061 strain (46) was used for cloning and expression. Cells were grown on LB broth in the presence of 100 μg/ml ampicillin for positive selection.

Production and purification of recombinant enzymes

Cultures and protein extracts were prepared following previously published procedures (23) with two modifications: (i) cells from a 1-liter culture were disrupted in 40 ml of 50 mm MOPS, 1 mm CoCl₂, pH 7.2, and (ii) protein extracts were heated at 70 °C for 10 min. The purification consisted of three chromatographic steps. The first step was anion-exchange chromatography on Source 15Q resin (GE Healthcare, Tricorn 10/150 column) in 50 mm MOPS, 1 mm CoCl₂, pH 7.2. Elution was performed with a gradient step from 0 to 0.5 m NaCl for five column volumes (CV). Fractions containing the protein of interest (2 CV) were pooled, and (NH₄)₂SO₄ powder was added to a concentration of 1.5 m (NH₄)₂SO₄. The second chromatographic step was hydrophobic-interaction chromatography on Source 15Phe resin (GE Healthcare, XK 16/20 column) in 50 mm MOPS, 1.5 m (NH₄)₂SO₄, 1 mm CoCl₂, pH 7.2. Elution was performed with a gradient step from 1.5 to 0 m (NH₄)₂SO₄ for 5 CV. Fractions (1.5 CV) containing the protein of interest were pooled and concentrated using an Amicon Ultra-15 ultrafiltration unit with 30-kDa cutoff (Merck Millipore). The third step consisted of gel filtration on Superdex 200 resin (GE Healthcare, XK 16/70 column) in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, 1 mm CoCl₂, pH 7.2. The concentration of (NH₄)₂SO₄ had to be maintained at 0.5 m to avoid protein precipitation. Purified proteins were finally concentrated using an Amicon Ultra-15 ultrafiltration unit with 30-kDa cutoff. The presence and purity of the recombinant enzymes were checked throughout the purification procedure by SDS-PAGE and enzymatic assays with l-Leu-pNA as substrate (see below). Proteins were quantified by measuring the absorbance at 280 nm and applying the extinction coefficient of 18,910 m⁻¹ cm⁻¹. This purification protocol allowed the purification of about 10 mg of TmPep1050 from 1 liter of culture. The concentrations of purified TmPep1050 are given in Table S1. Molecular weights were determined by gel filtration on Superdex 200 resin (GE Healthcare, XK 16/70 column) and Superpose 6 10/300 GL (GE Healthcare) using 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2, as running buffer. The gel-filtration columns were calibrated using both a high-molecular-weight gel filtration calibration kit (GE Healthcare) and gel-filtration standard (Bio-Rad).

Enzymatic assays

Aminoacyl-pNA substrates were purchased from Bachem AG. Aminopeptidase (EC 3.4.11.1) activity was assayed as described previously (23) except that enzymatic reactions were stopped by adding 1 ml of 20% acetic acid to 1 ml of reaction mixture (200 μl of substrate in 10% methanol, 790 μl of 50 mm MOPS, pH 7.2, and 10 μl of enzyme at a concentration ranging from 10 to 50 nm depending on the substrate). For the determination of substrate specificity of TmPep1050_12-mer, aminopeptidase assays were carried out at 75 °C with an enzyme concentration of 30 nm and the appropriate amino acid-pNA substrate. All substrates were used at 2.5 mm except l-Gly-pNA (1.25 mm), l-Phe-pNA (0.75 mm), and l-His-pNA and l-Glu-pNA (0.5 mm). The effects of metal ions, pH, and temperature on the activity were studied as described previously (23). For the determination of kinetic parameters, assays were performed at 75 °C with an enzyme concentration of 10 nm for l-Leu-pNA and l-Ile-pNA and an enzyme concentration of 50 nm for l-Met-pNA. Reaction mixtures were supplemented with 250 μm CoCl₂. Kinetic parameters (k_cat, K_m, and k_cat/K_m) were determined from the initial reaction rates using Lineweaver–Burk linearization of the Michaelis–Menten equation. Activation energies were calculated from the slope of the trend line obtained by plotting the logarithm of the specific activity versus the inverse of the temperature. For thermostability assays, TmPep1050_12-mer was diluted to 1 μm in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, 1 mm CoCl₂, pH 7.2, and it was incubated at either 75 or 95 °C. At various time intervals, the activity was measured at the incubation temperature (75 or 95 °C) by diluting the enzyme to 10 nm in 1 ml of reaction mixture containing 2.5 mm l-Leu-pNA as substrate.

Cobalt binding assays

A 1 mm TmPep1050_12-mer sample was diluted in 10 volumes of 2.1 m malic acid, pH 7.0, and concentrated back to 1 volume using an Amicon Ultra-15 ultrafiltration unit with a 30-kDa cutoff. The malic acid–treated sample was dialyzed four times against 100 volumes of 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2 using SnakeSkin^TM dialysis tubing with a 3.5-kDa cutoff (Thermo Fisher Scientific). To monitor metal ion removal, the specific activity was measured with l-Leu-pNA as substrate. Cobalt binding assays were performed by incubating 100 μl of 20 μm cobalt-depleted TmPep1050_12-mer with CoCl₂ at a concentration ranging from 0 to 2560 μm in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2, for 24 h at 75 °C. After incubation, the specific activity was measured using 10 nm enzyme and 2.5 mm l-Leu-pNA as substrate without added cobalt in the reaction mixture. Other metal ions were tested following this procedure to identify the metal cofactor of TmPep1050_12-mer.

In parallel, cobalt concentration was determined using Amplex UltraRed (Thermo Fisher Scientific), a fluorescent probe that specifically binds cobalt at high pH. The protocol established by Tsai and Lin (47) was adapted to fit a 384-well black microplate (Corning). Fluorescence was measured on a SpectraMax 5 (Molecular Devices) with excitation wavelength set on 495 nm and emission wavelength set on 570 nm. Prior to cobalt ion quantification, samples were diluted twice in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2. In addition to total cobalt concentration, unbound cobalt was quantified after filtering samples using an Amicon Ultra-0.5 ultrafiltration unit with a 30-kDa cutoff. The affinity constant K_d was determined from Scatchard plot data representation.

To study the reassociation of dimers into dodecamers, 100 μl of 50 μm TmPep1050_2-mer was incubated with Co²⁺ at a concentration ranging from 0 to 5 mm for 30 min at 75 °C. The oligomers were detected and quantified by size-exclusion chromatography using Superdex 200 resin (GE Healthcare Life Sciences, XK16/20 column). 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2, buffer was used for this assay.

Thermal shift assay

SYPRO Orange^TM (Thermo Fisher Scientific) was diluted 1:125 in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2. The fluorescent probe was mixed with protein samples conditioned in 50 mm MOPS, 0.5 m (NH₄)₂SO₄, pH 7.2, and Co²⁺ at a concentration ranging from 0 to 2560 μm. The working SYPRO Orange dilution was 1:1000, and the protein concentration was 20 μm for a reaction volume of 20 μl. Thermal shift assays were performed in a 96-well plate on a StepOnePlus^TM Real-Time PCR System (Thermo Fisher Scientific). Fluorescence curves were treated with StepOne^TM software.

Native MS

Samples for native MS were transferred into 20 mm ammonium acetate, pH 7.2. This was done using Zeba 7-kDa desalting columns (Thermo Fisher Scientific). If further desalting was needed, Bio-Spin P-6 gel columns (Bio-Rad) were used. The protein samples were diluted in 100 mm ammonium acetate, pH 7.2, to a working concentration of 5 μm unless stated otherwise. In house–prepared borosilicate gold-coated needles filled with 2–3 μl of sample were used to introduce the protein into the gas phase using nanoelectrospray ionization. The spectra were recorded in positive ion mode on a traveling-wave ion mobility Q-TOF instrument (Synapt G2 HDMS, Waters). Different settings were tuned to optimize sample measurement. The most important settings applied during the measurements, unless stated differently in the figure legends, were 20-V sampling cone, 10-V trap collision energy, and pressures of 8.30 e⁻³ and 5.31 e⁻² millibar for the source and trap, respectively.

Crystallization

TmPep1050_12-mer, TmPep1050_2-mer, TmPep1050_H60A, and TmPep1050_H307A were crystallized using the hanging-drop vapor diffusion method at 292 K in EasyXtal Tool plates (Qiagen). Drops contained 2 μl of recombinant enzyme mixed with 2 μl of well buffer. Crystallization conditions are described in Table S1. One cycle of seeding was necessary to get monocrystals of each species.

Structure determination and analysis

For apo-TmPep1050_12-mer, diffraction data were collected on the FIP-BM30a beamline at the European Synchrotron Radiation Facility (Grenoble, France) (48, 49). For TmPep1050_12-mer, TmPep1050_2-mer, TmPep1050_H60A, and TmPep1050_H307A, diffraction data were collected on Proxima 2 beamline at SOLEIL (Saint-Aubin, France). The data collection and refinement statistics are presented in Table 4. Diffraction data were processed using the XDS program package (50). Molecular replacement and model building were performed using PHENIX software package v.1.10.1–2155 (51). The initial solution of TmPep1050_12-mer was determined by molecular replacement with MR-Rosetta using the coordinates of YpdE of S. flexneri (PDB code 1YLO) as the search model (translation-function Z-score value, 23.9; log-likelihood gain, 5150). For TmPep1050_2-mer, TmPep1050_H60A, and TmPep1050_H307A, molecular replacement was achieved with Phaser-MR using the coordinates of TmPep1050_12-mer as search model. The models were built using phenix.autobuild. Iterative manual building was done in Coot (52). Multiple rounds of refinement were performed using phenix.refine. Model stereochemical quality was assessed using MolProbity (53). Protein structures were analyzed with PDBePisa (54), Arpeggio (55), Rosetta pK_a protocol (56, 57), Advanced Poisson–Boltzmann Solver (APBS) (58), and PyMOL Molecular Graphics System version 2.2 (Schrödinger, LLC).

Table 4.

Data collection and refinement statistics

Values in parentheses are for the highest-resolution shell. ESRF, European Synchrotron Radiation Facility; ASU, asymmetric unit.

	Apo-TmPep105012-mer	TmPep105012-mer	TmPep10502-mer	TmPep1050H60A	TmPep1050H307A
Data collection
Temperature (K)	100	100	100	100	100
Radiation source	ESRF BM30a	SOLEIL Proxima 2	SOLEIL Proxima 2	SOLEIL Proxima 2	SOLEIL Proxima 2
Wavelength (Å)	0.9797	0.9801	0.9800	0.9801	0.9801
Detector	ADSC QUANTUM 315r	DECTRIS EIGER X 9M	DECTRIS EIGER X 9M	DECTRIS EIGER X 9M	DECTRIS EIGER X 9M
Rotation range (°)	0.37	0.10	0.20	0.10	0.10
Exposure time (s)	20	0.025	0.025	0.025	0.025
Space group	P 1	H 3	C 2 2 21	C 2 2 21	P 1 21 1
Unit cell parameters
α, β, γ (°)	114.46, 91.71, 105.69	90.00, 90.00, 120.00	90.00, 90.00, 90.00	90.00, 90.00, 90.00	90.00, 110.51, 90.00
a, b, c (Å)	114.26, 114.57, 114.04	131.15, 131.15, 285.61	42.55, 114.71, 267.69	42.63, 114.22, 267.96	42.79, 138.65, 61.25
Resolution (Å)	44.05–2.20 (2.28–2.20)	47.60–1.70 (1.74–1.70)	48.25–2.00 (2.05–2.00)	43.46–1.84 (1.89–1.84)	40.11–1.75 (1.79–1.75)
Unique reflections	237,152	201,316	45,086	57,222	67,094
Rmerge (%)	8.9 (39.2)	8.2 (67.0)	9.5 (69.1)	8.7 (60.6)	5.6 (56.3)
Redundancy	3.2 (2.2)	10.34 (10.41)	13.2 (13.0)	12.0 (9.8)	6.7 (6.7)
〈I/σ〉	8.56 (2.22)	15.81 (2.81)	16.81 (3.46)	16.52 (2.63)	18.04 (2.38)
Completeness (%)	93.5 (84.8)	99.8 (97.6)	99.9 (99.2)	99.4 (91.7)	99.5 (97.2)
CC1/2 (%)	99.4 (81.1)	99.9 (87.1)	99.8 (90.0)	99.9 (90.0)	99.9 (88.1)
Refinement
Resolution	44.05–2.20	47.60–1.70	48.25–2.00	43.46–1.84	40.11–1.75
Reflections	237,090	201,316	45,076	57,213	67,094
Rfree set test count	11,854	9,715	2,254	2,862	3,363
Rwork/Rfree	0.212/0.247	0.143/0.164	0.166/0.203	0.167/0.195	0.165/0.185
Protein molecules per ASU	12	4	2	2	2
VM (Å3/Da)	2.98	3.27	2.26	2.26	2.36
Solvent content (%)	58.7	62.4	45.6	45.6	47.9
Protein/solvent atoms	29,969/2,223	10,759/1,474	4,610/362	4,730/500	4,621/335
r.m.s.d. bond lengths (Å)	0.009	0.019	0.004	0.004	0.012
r.m.s.d. bond angles (°)	1.254	1.680	0.647	0.672	1.263
Average B-factors (Å2)	37.0	24.0	37.8	33.2	28.9
Favored/disallowed Ramachandran ϕ/ψ (%)	95.71/0.46	95.35/0.34	94.86/0.17	94.95/0.00	93.64/0.52
Twin law					h, −k, −h−l
PDB code	4P6Y	6NW5	5NE6	5NE7	5NE8

Author contributions

R. D., F. S., and L. D. conceptualization; R. D. and N. B. formal analysis; R. D. and N. B. validation; R. D., T. V. G., and J. V. D. investigation; R. D. and T. V. G. visualization; R. D. and N. B. methodology; R. D., J. V. D., and L. D. writing-original draft; R. D. and L. D. writing-review and editing; D. V. E. resources; D. V. E. software; F. S. and L. D. supervision; F. S. and L. D. project administration.