Crystal structures of Mycobacterium tuberculosis RecA and its complex with ADP-AlF(4): implications for decreased ATPase activity and molecular aggregation

Abstract

Sequencing of the complete genome of Mycobacterium tuberculosis, combined with the rapidly increasing need to improve tuberculosis management through better drugs and vaccines, has initiated extensive research on several key proteins from the pathogen. RecA, a ubiquitous multifunctional protein, is a key component of the processes of homologous genetic recombination and DNA repair. Structural knowledge of MtRecA is imperative for a full understanding of both these activities and any ensuing application. The crystal structure of MtRecA, presented here, has six molecules in the unit cell forming a 6(1) helical filament with a deep groove capable of binding DNA. The observed weakening in the higher order aggregation of filaments into bundles may have implications for recombination in mycobacteria. The structure of the complex reveals the atomic interactions of ADP-AlF(4), an ATP analogue, with the P-loop-containing binding pocket. The structures explain reduced levels of interactions of MtRecA with ATP, despite sharing the same fold, topology and high sequence similarity with EcRecA. The formation of a helical filament with a deep groove appears to be an inherent property of MtRecA. The histidine in loop L1 appears to be positioned appropriately for DNA interaction.

Figure 1

Overall structure of MtRecA and its comparison with EcRecA. (A) A schematic representation using MOLSCRIPT (53) and Raster3D (54) of the crystal structure of MtRecA. N, M and C shown in green, red and blue, respectively, refer to the three domains. Phosphate groups are shown in ball and stick representation. α-Helices are represented by cylinders. β-Strands are represented by arrows and shown in a lighter shade in each domain as compared to the α-helices. (B) Pairwise sequence alignment (55,56) of EcRecA and MtRecA. The figure was prepared using BOXSHADE (57), showing identical regions highlighted on a red background, while regions with conservative substitutions are highlighted in yellow. Schematic depiction of secondary structural elements (showing spirals for helices and arrows for β-strands) were incorporated into the alignment. (C) Deviations in C_α positions of the MtRecA structure upon superposition on the EcRecA structure. This plot was prepared using GNUPLOT (58).

Figure 2

Domain and filament interfaces in MtRecA. (A) Superposition of EcRecA (grey) on MtRecA (black) using all C_α atoms. Only those segments that contribute to the small rearrangements in domain orientation are shown. Some side chains directly involved in interface interactions are shown in stick representation and labelled. The large shift in residue 35 (Met35→Gln) is particularly noteworthy. (B) Addition to the α/β/α layer upon filament formation leading to generation of a hybrid supersecondary structural element. α-Helices and β-strands are denoted by cylinders and arrows, respectively, and labelled according to their occurrence in the sequence. One molecule is shown in grey and the other in black. This figure was prepared using MOLSCRIPT and Raster3D.

Figure 3

Filament formation by RecA protein. Surface representations of filaments of (A) MtRecA, (B) EcRecA, (C) mt95 and (D) ec95, using GRASP (59). They are colour coded using atomic charges, red indicating a negative charge and blue a positive charge. (E) and (F) depict the filaments in (A) and (B), respectively, viewed down the z-axis. The central groove in the filament where DNA is expected to bind can be clearly seen.

Figure 4

Higher order association of filaments into bundles. (A) Two filaments of MtRecA are shown side by side, with each molecule in a filament in a different colour. This figure was prepared using INSIGHTII (24). The interface between the filaments involved in bundle formation is boxed and is shown in detail in (B). The corresponding interface in EcRecA is also shown for comparison in (C). In both (B) and (C) the interacting molecule of the first filament is shown in green and that of the second in yellow. Hydrogen bonds are indicated by broken lines. This figure was prepared using MOLSCRIPT and Raster3D.

Figure 5

Protein–nucleotide interactions in the MtRecA–ADP–AlF₄ complex. (A) Difference (F_o – F_c) electron density map contoured at 2.5σ, shown in red, into which ADP–AlF₄ has been fitted and refined. Tyr103, which makes stacking interactions with the adenine base can also be seen. This figure was prepared using BOBSCRIPT (60) and Raster3D. (B) Movement of the P-loop in MtRecA (orange), indicating widening of the binding pocket. The P-loop in EcRecA (green) is shown for comparison. ADP–AlF₄ in MtRecA is shown in ball and stick representation. (C) Surface representation of the nucleotide-binding pocket in MtRecA, using GRASP. The nucleotide molecule in it is shown in ball and stick representation. (D) The P (orange), B (purple) and S (blue) regions of the nucleotide-binding pocket showing side chains hydrogen bonding with ADP–AlF₄. (E) Schematic diagram showing interactions of ADP–AlF₄ with MtRecA. (C)–(E) were prepared using MOLSCRIPT and Raster3D.