Identification and mechanistic analysis of an inhibitor of the CorC Mg2+ transporter.

The CorC/CNNM family of Na+-dependent Mg2+ transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. CorC and CNNM have attracted interest as therapeutic targets, whereas inhibitors targeting the ATP-binding site have not been identified. Here, we performed a virtual screening of CorC by targeting its ATP-binding site, identified a compound named IGN95a with inhibitory effects on ATP binding and Mg2+ export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more toward the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift.

Introduction

CorC, a prokaryotic member of the CorC/CNNM family of proteins, is involved in Mg2+ transport (Akanuma et al., 2014; Armitano et al., 2016; Hmiel et al., 1989; Lee et al., 2019; Trachsel et al., 2019). In the pathogenic bacterium Staphylococcus aureus, CorC confers resistance to the high concentrations of Mg2+ ions in the infected host, increasing the pathogenicity of the bacterium (Armitano et al., 2016; Trachsel et al., 2019). Upon exposure to ribosome-targeting antibiotics, the expression of CorC is upregulated in the L22∗ strain of Bacillus subtilis to enhance Mg2+ flux for resistance to antibiotics (Lee et al., 2019). Furthermore, in humans, CNNM proteins, eukaryotic members of the CorC/CNNM family of proteins, are involved in a number of biological events, such as body absorption/reabsorption of Mg2+, hypertension, genetic disorders, and tumor progression (Funato et al., 2014, 2017; Hardy et al., 2015; Kostantin et al., 2016; Parry et al., 2009; Polok et al., 2009; Stuiver et al., 2011; Yamazaki et al., 2013). Therefore, CorC and CNNM are possible targets for novel antibiotics and drugs for treating various diseases, such as cancer. Of note, whether eukaryotic CNNM proteins directly transport Mg2+ ions or indirectly regulate Mg2+ transport remains controversial (Arjona and de Baaij, 2018; Funato et al., 2018a, b).

CorC/CNNM family proteins share a conserved transmembrane (TM) DUF21 domain and a cytoplasmic cystathionine-beta-synthase (CBS) domain with the regulatory ATP-binding motif (Armitano et al., 2016; de Baaij et al., 2012; Funato and Miki, 2019; Hmiel et al., 1989). The recently determined structures of the TM and CBS domains of the CorC Na+/Mg2+ antiporter from Thermus parvatiensis (TpCorC) revealed the mechanisms of Mg2+ and ATP binding, respectively (Huang et al., 2021). Subsequently, the crystal structures of CorC from Methanoculleus thermophiles, containing the TM and CBS domains, were also reported on bioRxiv (Chen et al., 2021). Furthermore, multiple structures containing the CBS domain of CNNM proteins have also been reported thus far (Chen et al., 2018, 2020; Corral-Rodriguez et al., 2014; Gimenez-Mascarell et al., 2019; Giménez-Mascarell et al., 2017, 2019; Gulerez et al., 2016; Zhang et al., 2017).

The Na+ gradient is implicated as a driving force for Mg2+ export from CorC and CNNM (Huang et al., 2021; Yamazaki et al., 2013), whereas ATP binding to the CBS domain of CorC and CNNM proteins is essential for the regulation of Mg2+ efflux activities (Chen et al., 2020; Hirata et al., 2014; Huang et al., 2021). Therefore, chemical compounds targeting the CBS domain of the CorC/CNNM family proteins, especially their ATP-binding site, could be exploited for therapeutic interventions against various diseases, but such compounds have not yet been identified, hindering the development and optimization of chemical compounds targeting the CorC and CNNM proteins. Furthermore, how ATP binding to CorC and CNNM proteins modulates their transport activity also remains unclear.

In this work, based on the ATP-bound structure of the CorC CBS domain, we performed virtual screening and functional assays to identify chemical compounds targeting the ATP-binding site of the CorC CBS domain and identified the chemical compound with inhibitory effects on ATP binding and Mg2+ export. Structural and biochemical analyses provided mechanistic insights for further design and optimization of chemical compounds targeting the ATP-binding site of CorC as well as mechanistic insights into how ATP and chemical compounds modulate the transport activity of CorC.

Results

Virtual screening

A computational strategy was applied to find potential active compounds against CorC (Figure 1). First, 6,412 compounds designed in-house were docked to the ATP-binding site in the CorC CBS domain structure (PDB ID: 7CFI). Then, 12 docked compounds among the compounds with the top 30 2D similarities with ligand efficiencies better than −0.40 kcal/mol, which interacted with at least three residues in the pocket via hydrogen bonds and π-π interactions, were chosen for further molecular mechanics with generalized Born and surface area solvation (MM/GBSA) calculations (Figure 2 and Table 1). As shown in Table 2, three available compounds (IGN95a, IGN23, and IGN19) with binding free energies greater than −10 kcal/mol were selected for the bioassay.

Figure 1

Computational strategy to identify active compounds

Molecular docking, 2D similarity analysis, atom efficiency analysis, and MM/GBSA calculations were combined to identify active compounds against CorC.

Figure 2

Compound selection from 2D similarity and atom efficiency analysis

The top 30 compounds with the highest 2D similarity to ATP were selected first. Among them, 12 compounds with atom efficiencies better than −0.40 (colored red) were chosen for further MM/GBSA calculations.

Table 1

Twelve compounds selected are ranked by atom efficiency

Molecule name	Chemical structure	Docking score (kcal/mol)	2D similarity	Atom efficiency	Interaction diagrams in docked models
IGN52		−10.22	0.59	−0.54
IGN23		−8.61	0.26	−0.54
DWF-17		−9.83	0.71	−0.52
IGN95a		−8.65	0.26	−0.51
GZ53		−11.34	0.26	−0.45
IGN19		−11.33	0.59	−0.45
SFS7		−9.58	0.26	−0.44
130354		−9.59	0.27	−0.42
130348		−12.32	0.26	−0.40
CS90		−10.75	0.26	−0.40
MR18		−10.35	0.27	−0.40
100628		−9.26	0.40	−0.40

Table 2

The binding free energies of 12 compounds to TpCorC calculated by MM/GBSA

Molecule name	Evdw	Eele	Egb	Esurf	ΔH	-TΔS	ΔGcal
IGN95a	−28.51±0.31	196.87±2.60	−194.94±2.30	−4.17±0.02	−30.75±0.54	16.87±0.95	−13.88±1.49
IGN23	−27.90±0.28	198.84±4.89	−197.55±4.26	−3.93±0.02	−30.54±0.70	17.17±2.16	−13.37±2.86
GZ53	−41.53±0.27	−41.91±1.63	53.06±1.49	−5.75±0.02	−36.13±0.38	24.63±0.84	−11.5±1.22
IGN19	−35.94±0.37	−46.84±1.46	52.22±1.28	−4.64±0.03	−35.21±0.68	24.91±3.37	−10.3±4.05
100628	−35.18±0.25	−26.07±0.63	35.57±0.56	−4.46±0.02	−30.14±0.26	20.06±0.46	−10.08±0.72
SFS7	−33.49±0.30	9.04±1.33	9.05±1.23	−4.52±0.03	−19.93±0.33	13.64±2.22	−6.29±2.55
IGN52	−28.90±0.25	−36.99±1.25	44.28±1.10	−3.89±0.02	−25.49±0.29	20.73±3.26	−4.76±3.55
CS90	−34.96±0.24	−33.75±0.62	50.82±0.55	−4.75±0.03	−22.64±0.31	20.02±1.02	−2.62±1.33
DWF-17	−31.65±0.25	−22.05±1.34	33.86±1.02	−4.05±0.03	−23.89±0.58	21.87±3.11	−2.02±3.69
MR18	−28.10±0.48	−68.92±1.57	73.54±1.29	−5.15±0.04	−28.64±0.53	27.53±0.82	−1.11±1.35
130348	−11.48±0.57	−1,684.72±5.22	1,668.17±5.27	−4.54±0.03	−32.58±0.60	31.56±1.38	−1.02±1.98
130354	−7.01±0.32	−940.06±2.86	928.9±2.68	−2.49±0.03	−20.68±0.50	19.92±0.73	−0.76±1.23

Evdw: the van der Waals energy contribution; Eele: the electrostatic energy contribution; Egb: electrostatic contribution to the solvation free energy; Esurf: non-polar energy contribution to the solvation free energy; ΔH = Evdw + Eele + Egb + Esurf; TΔS: the entropy changes; ΔGcal: the binding free energy between each compound and TpCorC.

In vitro screening

We then performed fluorescent ATP-based binding assays with the chemical compound candidates from the virtual screening. We employed 2′(3′)-O-(N-methylanthraniloyl)adenosine 5′-triphosphate (mant-ATP) for the binding assay and measured fluorescence resonance energy transfer (FRET) from endogenous Trp residues to the bound mant-fluorophore (Göttle et al., 2007; Ni et al., 2000).

The FRET data showed that mant-ATP was bound to the purified CorC CBS domain with a Kd of 1.36 ± 0.10 μM (Figure 3A). For validation of this method, we performed a competitive binding assay with ATP (Figure 3B). The addition of ATP at the respective concentrations yielded an IC50 value of 0.57 ± 0.08 μM, which is comparable with the previously reported Kd value of the CorC CBS domain for ATP obtained by isothermal titration calorimetry (ITC) (Huang et al., 2021).

Figure 3

Mant-ATP binding assay

(A) Saturation of mant-ATP binding to the CorC CBS domain (1 μM) detected by FRET. Data are expressed as the mean ± SE. R2 = 0.9989, n = 3.

(B) ATP-based inhibition test of the binding of the CorC CBS domain (1 μM) and mant-ATP (1 μM) using FRET. Data are expressed as the mean ± SE. R2 = 0.9894, n = 3.

(C and D) Mant-ATP binding inhibition by chemical compounds. (C) Data are expressed as dots. n = 2. Each chemical compound was added at 0.25 mM. (D) Data are expressed as the mean ± SE. n = 6. IGN95a was added at 2 mM.

Finally, from the mant-ATP-based screening of chemical compound candidates from the virtual screening, we identified IGN95a, an adenine analog, as the compound that most potently inhibited mant-ATP binding (Figures 3C and 3D).

Characterization of IGN95a

We further characterized IGN95a (Figure 4). First, the ITC experiment confirmed IGN95a binding to the CorC CBS domain with a Kd value of 47.0 μM (Figures 4A and Table S1). Notably, we did not exclude the possibility that the estimated Kd might be affected by the partially unremoved ATP in the purified protein to some extent given that the wild-type CBS domain protein exhibits high affinity for ATP (∼500 nM) (Huang et al., 2021).

Figure 4

Functional characterization of IGN95a

(A) ITC data of the TpCorC CBS domain with IGN95a. The raw ITC data and profiles are shown. Measurements were repeated twice, and similar results were obtained.

(B) Mg2+ export assay of CorC-expressing cells treated with IGN95a. Line graph: time course of mean relative fluorescent intensities. Mg2+ was depleted at the time point indicated with an arrowhead. Bar graph: relative fluorescence intensities after Mg2+ depletion at 5 min. The data are shown as mean ± SEM (Empty: n = 10, WT without IGN95a: n = 18, WT with 100 μM IGN95a: n = 19, and WT with 1,000 μM IGN95a: n = 30).

(C and D) ITC data of the CBS domain of CNNM2 (C) and CNNM4 (D) with IGN95a.

We then tested the effects of IGN95a on the Mg2+ export activity of CorC (Figure 4B). We performed Mg2+ export activity assay with Magnesium Green, a fluorescent Mg2+ indicator dye that has a wide dynamic range and is well suited for sensitively monitoring the change in Mg2+ levels in time-lapse analyses. We employed the human embryonic kidney 293 (HEK293) cell line stably expressing CorC at the cell surface, as it was employed for the previous structure-based mutational analysis of CorC (Huang et al., 2021). The intensity of the fluorescent signal in the control cells expressing CorC soaked with a buffer containing only 1% DMSO decreased after the removal of Mg2+ ions from the bath solution, whereas we observed inhibitory effects of IGN95a on the Mg2+ export activity of CorC after soaking with IGN95a in 1% DMSO buffer with HEK293 cells expressing CorC (Figure 4B). These results show that IGN95a acts as an inhibitor of both ATP binding and Mg2+ export, and also indicates that IGN95a can permeate the mammalian cell membrane. Consistently, the computationally calculated miLogP value of 1.26 also suggested the cell membrane permeability of IGN95a. Thus, considering the miLogP value and mammalian cell permeability, we hypothesize that IGN95a can also likely permeate the bacterial cell membrane.

Furthermore, we tested IGN95a binding to the CBS domain of human CNNM2 and CNNM4 (Figures 4C and 4D). In the ITC experiments, IGN95a showed weak binding to CNNM2 and CNNM4. The exact Kd values could not be estimated because the titrations were not completed (>1,000 μM for the CBS domain of CNNM2, >500 μM for the CBS domain of CNNM4). To estimate the exact Kd values, other methods with a higher dynamic range of dissociation constants, such as microscale thermophoresis, can be potentially applicable.

Overall, these results suggest the specificity of IGN95a against CorC compared with CNNM family proteins. Given that CNNM2 and CNNM4 mutations are associated with human genetic diseases, including dominant hypomagnesemia (Stuiver et al., 2011) and Jalili syndrome (Parry et al., 2009; Polok et al., 2009), the specificity of IGN95a against CorC compared with CNNM family proteins may be beneficial for future optimization to avoid side effects in vivo.

Inhibitor-bound structure

To understand the molecular interactions between the CorC CBS domain and IGN95a, we performed co-crystallization of the TpCorC CBS domain with IGN95a.

As the wild-type CBS domain protein has a very high affinity for ATP, it was difficult to completely remove endogenous ATP during purification, which is not ideal for co-crystallization with IGN95a. Therefore, we employed the T336I mutant with weaker affinity for ATP (Huang et al., 2021) because mutation at Thr336 was relatively unlikely to affect IGN95a binding based on the initial docking model. Indeed, we successfully determined the crystal structure of the TpCorC CBS domain in complex with IGN95a (Figures 5A and S1).

Figure 5

IGN95a-bound structure of the CorC CBS domain

(A and B) Close-up views of the IGN95a-binding (A) and ATP-binding (B) sites in the CorC CBS domain. Ligands and the surrounding residues are shown in stick representation. Dashed lines indicate hydrogen bonds.

(C) 2D interaction diagram between IGN95a and the CorC CBS domain.

(D and E) ITC data of CorC CBS domain mutants with IGN95a. The raw ITC data and profiles are shown. Measurements were repeated twice, and similar results were obtained.

The adenine ring of IGN95a is recognized by the TpCorC domain (Figure 5A), similar to ATP (Figure 5B). One of the oxygen groups forms an additional hydrogen bond with the side chain of Ser256 (Figures 5A and 5C). In the ATP-bound structure, Thr336, Glu338, and Asp339 form multiple hydrogen bonds with ATP (Figure 5B), but the corresponding residues are not involved in direct interactions with IGN95a (Figure 5A).

The binding pose of IGN95a with CorC in the crystal structure is similar to the docking pose, including the same π-π interaction with Tyr255 (Figures S2A and S2B). To examine the stability of the binding of IGN95a to CorC, explicit-solvent molecular dynamics (MD) simulation of the crystal structure of the CorC CBS domain in complex with IGN95a was performed. The representative conformation of the largest clusters of the 300-ns trajectories for IGN95a have similar interactions with the key residues (Val235, Tyr255, and Arg257) in the crystal structure (Figures 5A and S3A). Compared with the initial structure, the root-mean-square deviations for IGN95a range from 0.2 Å to 1.5 Å (Figure S3B), and the key interactions exist during the 300-ns MD simulations (Figures S3C and S3D), implying that IGN95a is stable in the binding pocket.

To further verify the structure, we performed a binding assay of the ATP-binding site mutants Y255A (adenine ring) and T336I (ribose) using ITC. According to the structure, mutation at Tyr255 should severely affect IGN95a binding, whereas mutation at Thr336 should have a weaker impact on IGN95a binding (Figure 5A). The T336I mutant of the CBS domain exhibited a Kd value of 147.3 μM for IGN95a, whereas there were no detectable interactions between the Y255A mutant and IGN95a (Figures 5D and 5E, and Table S1), essentially supporting our structure. The POLDER-OMIT maps showed clear electron densities for most parts of IGN95a, except for the one for the ethyl group (Figure S1). Consistently, the ethyl group of IGN95a seems to exhibit no direct interactions with the CorC CBS domain, and would be exposed to the exterior of the ATP-binding pocket (Figure 5A). Thus, this part of IGN95a might be a promising modification target for further optimization of chemical compounds.

Effect of ATP and IGN95a on the structural equilibrium of the CorC TM domain

To gain insights into the ATP modulation and IGN95a inhibition mechanisms of CorC, we performed biochemical cross-linking experiments using the cross-linking mutant of TpCorC (Figure 6), which we established previously (Huang et al., 2021). The TM domain of TpCorC adopts an inward-facing conformation in the presence of Mg2+, with an inter-subunit distance of 7.1 Å between the Cβ atoms of Thr106 residues (Figure 6A), which is sufficiently close for chemical cross-linking through Cys residues. We previously generated the cross-linking mutant of TpCorC (T106C/C282A), where Cys282 was also mutated to remove an endogenous cysteine residue. In fact, previous cross-linking experiments with Cu2+ phenanthroline showed that the T106C pair of TpCorC formed a disulfide bond in the presence of Mg2+, as indicated by a strong band for the dimer on a non-reducing SDS-PAGE gel (Huang et al., 2021) (Figure 6B). Furthermore, whereas the addition of Na+ disrupted the cross-linked dimer, the replacement of Na+ with K+ resulted in bands for both the TpCorC monomer and dimer (Huang et al., 2021) (Figure 6B).

Figure 6

Inter-subunit chemical cross-linking of CorC with ATP and IGN95a

(A) Structure of the TpCorC TM domain dimer (PDB ID: 7CFF) in cartoon representation, viewed parallel to the cell membrane (left) and from the periplasmic side (right). The Thr106 residues are depicted in stick representation. Dashed lines show the Cα distances between the Thr106 residues within the dimer.

(B) Chemical cross-linking experiments of the inter-subunit cross-linking mutant (T106C/C282A) of TpCorC and its ATP-binding site mutant (T106C/T255A/C282A/T336I).

Intriguingly, the addition of ATP to the TpCorC cross-linking mutant in the absence of Na+ and Mg2+ led to a stronger dimer band than that of the ATP-free sample in the absence of Na+ and Mg2+ (Figure 6B), whereas we did not see such a shift upon the addition of IGN95a (Figure 6B). Based on this result, we hypothesized that ATP binding to the TpCorC CBS domain affects the conformational equilibrium of the TM domain toward more inward-facing conformations.

To further verify this hypothesis, we generated an ATP-binding site mutant of the TpCorC cross-linking construct (T106C/C282A/Y255A/T336I) for chemical cross-linking experiments (Figure 6B). Mutations in Y255A/T336I are known to abolish the ATP-binding activity of TpCorC as well as lower Mg2+ export activity (Huang et al., 2021). As expected, the addition of ATP to this mutant did not lead to a stronger dimer band than that of the original cross-linking mutant (Figure 6B), further supporting our hypothesis regarding the effect of ATP on TpCorC. Notably, because the TpCorC CBS domain has no ATP hydrolysis activity (Huang et al., 2021), ATP binding would be sufficient to affect the conformational equilibrium of the TM domain.

Discussion

In this work, we performed virtual and in vitro screening of CorC by targeting its ATP-binding site and identified a chemical compound, IGN95a, with inhibitory effects on both the ATP binding and Mg2+ export activities of CorC (Figures 1, 2, 3, and 4). Co-crystallization of the CorC ATP-binding domain with IGN95a and associated MD simulations provided structural insights for the further development and optimization of chemical compounds for the CorC ATP-binding site (Figure 5). Finally, chemical cross-linking experiments indicate that ATP binding to the CorC CBS domain shifts the conformational equilibrium of its TM domain toward more inward-facing conformations, whereas IGN95a, which occupies the ATP-binding site, does not have such an effect (Figure 6). Based on these results, we discuss the mechanisms of action of ATP and IGN95a on CorC (Figures 7 and 8)

Figure 7

Structural comparisons of the apo, ATP-bound, and IGN95a-bound structures of the CorC CBS domain

(A) Superposition of the IGN95a-bound CBS domain structure (yellow) onto the apo structure (red).

(B) Superposition of the ATP-bound CBS domain structure (cyan) onto the IGN95a-bound structure (yellow). Both subunits in the dimer are shown, and the neighboring subunit of the dimer is colored gray. Both the overall structure and close-up view of the region near the exterior of the ATP-binding site are shown. Black arrows indicate the structural changes between two conformations.

Figure 8

Proposed CorC regulation mechanisms by ATP and IGN95a

(A–C) Schematic diagrams of the conformational equilibrium of the CorC in the apo (A), ATP-bound (B), and IGN95a-bound (C) forms. ATP binding induces structural changes in the cytoplasmic domain at the interface between the TM and cytoplasmic domains, which shifts the conformational equilibrium of the transmembrane domain more toward the inward-facing state (A and B). In contrast, IGN95a binding inhibits the binding of ATP in a competitive manner and inhibits the associated structural changes, which in turn prevents the adjustment of the conformational equilibrium of the TM domain (C). Mg2+ and Na+ ions are shown as purple and orange spheres, respectively. Black arrows indicate the metal transport directions.

First, a comparison of the apo, IGN95a-bound, and ATP-bound structures of the CBS domain suggests that the IGN95a-bound structure is more similar to the apo structure than to the ATP-bound structure (Figure 7). In the ATP-bound structure, the helix region on the exterior of the ATP-binding site moves slightly outward from the pocket, mainly via its contact with the ribose moiety of ATP (Figure 7B). In contrast, IGN95a binding did not induce such movement (Figure 7A). Previous chemical cross-linking experiments and MD simulations suggested that this region, undergoing this ATP-dependent structural change, is located at the interface between the TM and CBS domains (Huang et al., 2021) (Figure S4). Consistent with this, our chemical cross-linking experiments indicated that ATP binding to the CorC CBS domain affects the conformational equilibrium of the TM domain, shifting it toward more inward-facing conformations (Figures 6 and 8), which would be more favorable for attraction of intracellular Mg2+ to the Mg2+-binding pocket in the TM domain. As the transport activity of CorC is lower in the absence of ATP binding (Huang et al., 2021), the ATP-dependent structural change in the CBS domain might be important for CorC to properly maintain its transport activity by adjusting the structural equilibrium of CorC toward a more inward-facing state through the contacts between the TM and CBS domains (Figures 8A and 8B). Notably, the binding of ATP to the CBS domain of the ClC-1 Cl− channel also affects its transport activity through the domain interface between the TM and CBS domains (Wang et al., 2019). In contrast, the binding of IGN95a to the ATP-binding site does not induce the ATP-dependent conformational change of the CBS domain at the interface between the TM and CBS domains (Figures 7, 8C, and S4). Considering the affinity of the CorC CBS domain for ATP (Huang et al., 2021) and the cytoplasmic ATP concentration (∼1 mM) (Beis and Newsholme, 1975; Yaginuma et al., 2014), CorC would be mostly in the ATP-bound form in vivo. Thus, the addition of IGN95a lowers the transport activity of CorC (Figure 4B), probably because IGN95a binding inhibits the binding of ATP in a competitive manner (Figure 5) and inhibits the associated structural changes (Figure 7), which in turn prevents the adjustment of the conformational equilibrium of the TM domain (Figures 6 and 8).

Limitations of the study

Notably, whether eukaryotic CNNM proteins directly transport Mg2+ ions or regulate Mg2+ transport remains controversial (Arjona and de Baaij, 2018; Funato et al., 2018a, 2018b). Given that this study focuses on bacterial CorC, this controversy does not affect our main conclusion. To settle the controversy, additional Mg2+ transport assays of eukaryotic CNNM proteins, particularly purified proteins in liposomes, should be performed, but these experiments are beyond the scope of this study. Furthermore, whereas IGN95a inhibited Mg2+ export activities of CorC in HEK293 cells, further characterization on Mg2+ export activities, particularly experiments using the bacterial system, are also required in the future.

Overall, our results provide not only structural insights for the further design and optimization of chemical compounds targeting the ATP-binding site of CorC but also mechanistic insights into how ATP and chemical compounds modulate the transport activity of CorC.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Motoyuki (hattorim@fudan.edu.cn).

Materials availability

Plasmids and other materials generated in this study can be requested from the lead contact, Motoyuki Hattori (hattorim@fudan.edu.cn).

Data and code availability

The atomic coordinates and structural factors for the structure of the CorC CBS domain in the IGN95a-bound form have been deposited in the Protein DataBank under accession code 7CFK.

Methods

All methods can be found in the accompanying transparent methods supplemental file.