Journal:Acta Cryst F:S2053230X19004618

Novel T9 loop interaction of Filamenting Temperature-sensitive mutant Z from Mycobacterium tuberculosis

E. O. Lazo, J. Jakoncic, S. RoyChowdhury, D. Awasthi, I. Ojima ^[1]

Molecular Tour
As of 2017, tuberculosis has infected 1.7 billion people (23% of the world's population) and has caused 10 million deaths. Mycobacterium tuberculosis (Mtb) is quickly evolving, and new strains are classified as multi-drug resistant. Thus, the development and discovery of new drugs to combat Mtb is vital to combat the drug-resistant strains. Filamenting temperature-sensitive mutant Z (FtsZ), an important protein involved in cell-division is key for the survival of Mtb. Here, we have solved the crystal structure of MtbFtsZ that exhibit an inter-subunit that plays a biological role in the GTPase activity of MtbFtsZ and have elucidated a novel conformation, involving the T9 loop and the nucleotide binding pocket that breaks up the GTPase active site. This novel conformation can serve as basis for the development of the novel drugs to combat tuberculosis.

(5v68). Protomers A and C shown in blue, protomer B shown in gray, protomers D and F shown in red, protomer E shown in cyan, GDP are represented by orange spheres, and PO4 by green spheres. . Same color scheme as in previous scene.

Comparison of other MtbFtsZ structures with trimer ABC from 5v68 reveals that protomers A and B superimposed well but the conformation of protomer C is vastly different. . Nucleotide for both is GDP. Color scheme is as follows, 5v68 blue, 4kwe yellow, nucleotides orange spheres, Glu231 green spheres, phosphate red spheres. . The trimer ABC from 5v68 is to a curved MtbFtsZ protofilament (PDB 4kwe). Based on an r.m.s.d. of 1.2Å between protomers AB of 5v68 and protomers CB of 4kwe, protomers A and B from 5v68 are similar with protomers C and B of 4kwe. .

. Color scheme is as follows, 5v68 blue, 2q1y green, nucleotides orange spheres, Glu231 green spheres, phosphate red spheres. . To generate this trimer (2q1y), A1 A2 A3, the crystal symmetry molecules neighboring chain A2 that exhibit the inter-subunit interface were used. Only chains A1 and A2 were used for the superimposition. Again, protomers A and B from present study are similar with the top two protomers A1 and A2 from 2q1y with an r.m.s.d of 2.1Å. is approximately 63Å.

. Color scheme is as follows, 4kwe yellow, 2q1y green, nucleotides orange spheres, Glu231 green spheres. . Superimposition of 4kwe and 2q1y reveals an r.m.s.d. of 2.8Å between the trimers and a 12.5Å , showing that these structures are relatively similar. The distances between Glu231 of 5v68 from protomer C is far greater than 12.5Å and the interaction with the middle protomers of 4kwe and 2q1y with protomer C of 5v68 involve the T9 loop residue Glu231, which is not the case in the other structures.

(gray). (orange). Helix H11 is shown in green. Helices η1, H7, and loop T6 are shown in blue. Switch I (T3 loop) is disordered surrounded by a cloud. Switch II (sH2) is shown in magenta. The top two protomers of all three structures (AB for 5v68, A1 A2 for 2q1y, and CB for 4kwe) all exhibit a similar inter-subunit interface. This interaction between protomers A and B of 5V68 involve the T6 and T7 loops, helices: H11, η1, and H7. Protomer A of our structure “sits” on the helices η1, H7 and loop T6 (all shown in blue) from protomer B. This brings the T7 loop’s (shown in red) residues of protomer A of 5v68 within 16Å of GDP from protomer B, forming the GTPase active site. The T3 loop is disordered or in its OFF position (surrounded by magenta cloud). Helix sH2 is also OFF because there is no hydrogen network (shown in magenta).

. Protomer B is shown in gray, the T9 loop in brown, T11 in cyan; and the N-terminal of protomer C is shown in green, C-terminal blue, helix H8 yellow, the T7 loop red, and the switches in magenta. . Glu231 from protomer B is shown in brown, while Glu274 from protomer is in gray. The residues in green are from protomer C.

References

1. ↑ Lazo EO, Jakoncic J, RoyChowdhury S, Awasthi D, Ojima I. Novel T9 loop conformation of filamenting temperature-sensitive mutant Z from Mycobacterium tuberculosis. Acta Crystallogr F Struct Biol Commun. 2019 May 1;75(Pt 5):359-367. doi:, 10.1107/S2053230X19004618. Epub 2019 Apr 24. PMID:31045565 doi:http://dx.doi.org/10.1107/S2053230X19004618