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. 2019 Aug 29;10(1):3906.
doi: 10.1038/s41467-019-11860-z.

Mycobacterial Dynamin-Like Protein IniA Mediates Membrane Fission

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Free PMC article

Mycobacterial Dynamin-Like Protein IniA Mediates Membrane Fission

Manfu Wang et al. Nat Commun. .
Free PMC article

Abstract

Mycobacterium tuberculosis infection remains a major threat to human health worldwide. Drug treatments against tuberculosis (TB) induce expression of several mycobacterial proteins, including IniA, but its structure and function remain poorly understood. Here, we report the structures of Mycobacterium smegmatis IniA in both the nucleotide-free and GTP-bound states. The structures reveal that IniA folds as a bacterial dynamin-like protein (BDLP) with a canonical GTPase domain followed by two helix-bundles (HBs), named Neck and Trunk. The distal end of its Trunk domain exists as a lipid-interacting (LI) loop, which binds to negatively charged lipids for membrane attachment. IniA does not form detectable nucleotide-dependent dimers in solution. However, lipid tethering indicates nucleotide-independent association of IniA on the membrane. IniA also deforms membranes and exhibits GTP-hydrolyzing dependent membrane fission. These results confirm the membrane remodeling activity of BDLP and suggest that IniA mediates TB drug-resistance through fission activity to maintain plasma membrane integrity.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Overall structures of IniA. a Scheme showing the domains of IniA in different colors. Secondary structure elements that form HBs are labeled. N, N-terminus; C, C-terminus. b Cartoon structure of GTP-bound IniA. As in (a), the GTPase, HB1/Neck, and HB2/Trunk domains and LI loop are colored pink, orange, green, and blue, respectively. GTP and tartaric acid (TA) are shown as red and magenta sticks. Mg2+ is represented by a green sphere. The disordered segment in the α9-loop is denoted by a dotted line. c Structural superposition of the apo IniA and GTP-bound IniA. GTP is shown as spheres. d Structural comparison of the apo structures between IniA and BDLP (PDB code: 2J69) (upper panel), and the GTP/GMPPNP-bound structures between IniA and BDLP (PDB code: 2W6D) (lower panel)
Fig. 2
Fig. 2
Analysis of the GTP-binding site. a The binding of GTP to IniA. GTP and nearby residues are shown as sticks. Mg2+ and water molecules (W) are represented by spheres. The 2FoFc electron density maps (contoured at 1.0 σ) of GTP, Mg2+, and water molecules are shown as wire mesh (blue). The hydrogen bond between S69 and R63 is indicated by the dashed line. b Structural alignment of nucleotides and conserved motifs between IniA and cyanobacteria BDLP. The dashed loop represents un-modeled fragment in BDLP. c Binding affinity of GDP/GMPPNP/GTP for wild-type (wt) IniA and the K46A mutant measured by ITC. The dissociation constant, Kd, is given below. The data are representative of at least three repetitions. d GTPase activity of IniA and different mutants. The activities were measured by the reaction rate at 0.5 mM GTP. Each bar is the mean and SD of three measurements. The source data of Fig. 2d are provided in the Source Data file
Fig. 3
Fig. 3
LI loop-mediated membrane association. a Residue conformation of the LI loop. Residues are shown as sticks, except that Gly is represented as a  sphere. Tartaric acid (TA) interacts with the main chain N atoms in the LI loop (indicated by the dashed lines). b Liposome (E. coli polar lipids containing 78 mol% PE, 12 mol% PG, 6 mol% CL, 4 mol% PA) flotation assay showing the effect of NaCl concentration (150 mM and 600 mM) on IniA membrane association. c As shown in (b), but testing the effect of different lipids (100 mol% PC; 50 mol% PE, 50 mol% PC; 80 mol% PE, 20 mol% PG; 80 mol% PE, 20 mol% CL; 80 mol% PE, 20 mol%PA; 78 mol% PE, 12 mol% PG, 6 mol% CL, 4 mol% PA) for recruiting IniA. d As shown in (b), but with mutants at the LI loop. Δ480–492, L480-K492 was replaced by the GGGGSGGGGS linker. e IniA(wt)-GFP fusion protein was expressed in M. smegmatis, and their localization determined by GFP (green) and compared with that of FM4–64 (red) inserted in the membrane by confocal microscopy. Scale bars, 2 µm. fh As shown in (e), the mutants IniA(Δ480–492)-GFP (f) and IniA(R488D)-GFP (g) and the control GFP (h) were also performed. Scale bars, 2 µm. The source data of Fig. 3b–d are provided in the Source Data file
Fig. 4
Fig. 4
Nucleotide-independent oligomerization. a The molecular masses of IniA (theoretical molecular mass 65.9 kDa) were determined from sedimentation coefficients measured by analytical ultracentrifugation in the absence or presence of 2 mM GDP/GMPPNP/GTP or 2 mM GDP, 5 mM AlCl3,10 mM NaF. The data are representative of at least three repetitions. b Liposomes (78 mol% PE, 12 mol% PG, 6 mol% CL, 4 mol% PA, final lipid concentration 2 mM) were mixed in various conditions, 2 μM IniA was added at initiation, and the absorbance was measured at 405 nm. The data are representative of at least three repetitions. c As shown in (b), the results were observed using a fluorescence microscope. Scale bar, 40 µm. d As shown in (b), 2 mM Mg2+ and 5 mM GTP (or 5 mM ATP as a control) were added after a period of reaction. The source data of Figs. 4a, b and d are provided in the Source Data file
Fig. 5
Fig. 5
GUV rupture by IniA. a In vitro GUVs (red) binding assays show different binding ability of 15 µM WT IniA, Δ480–492, and S50A/R63A mutants (green). Scale bar, 10 µm. b As shown in (a), WT IniA and S50A/R63A mutant with the time course after 0.5 mM GTP/GMPPNP incubation for 10 min, the arrows indicate the rupture sites. Scale bar, 10 µm. c GUVs treated with 15 µM WT IniA and S50A/R63A mutant in the presence of 0.5 mM GTP/GMPPNP during 45 min, untreated GUVs as a control. Scale bar, 10 µm. d The diagram shows the statistical results in (c), the number of GUVs seen at indicated time points was counted and shown as relative values to that at the start point. Each bar is the mean and SD of three measurements. The source data of Fig. 5d is provided in the Source Data file
Fig. 6
Fig. 6
Membrane fission by IniA. a SMrTs (red) were treated with wild-type IniA (IniA WT) and R488D mutant in the absence or presence of 5 mM GTP. Protein localization and tube cleavage were monitored using confocal microscopy. The arrows indicate the cleavage sites. Scale bar, 20 µm. b As shown in (a), but with wild-type IniA recorded along with the time course after GTP addition. The arrows indicate the cleavage sites. Scale bar, 20 µm
Fig. 7
Fig. 7
IniA-mediated isoniazid resistance in M. smegmatis. a Growth curves of wild-type M. smegmatis [Ms (WT)] and iniA-deleted strain [MsiniA::hyg)] in the presence or absence of 20 μg mL−1 isoniazid. b Growth curves of MsiniA::hyg) with overexpression of wild-type IniA (S9::iniA) in 20 μg mL−1 isoniazid, compared with no antibiotics or no IniA overexpression. c As shown in (b), overexpression of mutant IniA, including S50A/R63A, ΔHB2, R488D, and Δ480–492. Each bar is the mean and SD of three measurements. The source data of Fig. 7a–c are provided in the Source Data file

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References

    1. Vilcheze C, Jacobs WR., Jr. The mechanism of isoniazid killing: clarity through the scope of genetics. Annu Rev. Microbiol. 2007;61:35–50. doi: 10.1146/annurev.micro.61.111606.122346. - DOI - PubMed
    1. Belanger AE, et al. The embAB genes of Mycobacterium avium encode an arabinosyl transferase involved in cell wall arabinan biosynthesis that is the target for the antimycobacterial drug ethambutol. Proc. Natl Acad. Sci. USA. 1996;93:11919–11924. doi: 10.1073/pnas.93.21.11919. - DOI - PMC - PubMed
    1. Snider DE, Jr., et al. Standard therapy for tuberculosis 1985. Chest. 1985;87:117S–124S. doi: 10.1378/chest.87.2_Supplement.117S. - DOI - PubMed
    1. Alland D, et al. Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): the effect of isoniazid on gene expression in Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA. 1998;95:13227–13232. doi: 10.1073/pnas.95.22.13227. - DOI - PMC - PubMed
    1. Boot M, et al. iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum. J. Biol. Chem. 2016;291:19800–19812. doi: 10.1074/jbc.M116.724088. - DOI - PMC - PubMed

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