The ribosomal maturation factor P from Mycobacterium smegmatis facilitates the ribosomal biogenesis by binding to the small ribosomal protein S12

Abstract

The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N- and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections.

Keywords: MSMEG 2624; Mycobacterium smegmatis; RimP; RpsL; bacterial translation; crystal structure; mycobacteria; protein complex; protein-protein interaction; ribosomal biogenesis; ribosomal maturation factor P; ribosome assembly; small ribosomal protein S12; tuberculosis.

Figure 1.

The crystal structure of MSMEG_2624. A, the cartoon colors the structure of MS- MEG_2624 by secondary structure. B and C, comparison between the C-terminal domain of SP14.3 (B) and that of MSMEG_2624 (C). The additional α helix and the long connecting loop in MSMEG_2624 are highlighted in red. D, electron density of the interdomain linker, at 2.0σ level. E and F, residues participate in the interdomain interaction. Hydrogen bond interactions are highlighted using yellow dashed lines.

Figure 2.

Tandem affinity purification maps the MSMEG_2624–RpsL interaction. His-MS- MEG_2624 (A) or His-tagged individual MSMEG_2624 domains (B and C) and GST-RpsL were recombinantly co-expressed and purified through Ni²⁺ and then GST affinity purification. The fractions are labeled at the top of the corresponding lanes (FT, flow-through; GST-FT/wash/elution, GST affinity chromatography of fractions after the first His elution; GST-elution only, elution using the GST affinity chromatography without the Ni²⁺ affinity chromatography). Red arrows mark where the corresponding proteins are expected.

Figure 3.

Site-directed mutagenesis on the linker region of MSMEG_2624 and the effects on MSMEG_2624–RpsL interaction. A, crystal structure of the linker region of MSMEG_2624. Evolutionarily conserved residues are marked by an asterisk. B, pulldown assay determines the binding efficiency between GST-RpsL and His-tagged MSMEG_2624 and its mutants. Top, Western blot (anti-His antibody) detects the input amount of His-MSMEG_2624 after the first Ni²⁺ affinity chromatography. Middle, Coomassie staining detects the input amount of GST-RpsL after the second GST affinity chromatography. Bottom, Western blot (anti-His antibody) detects the amount of eluted His-MSMEG_2624 after the second GST affinity chromatography. Asterisks mark the evolutionarily conserved residues. Cyan rectangular boxes mark where the gels/blots are cropped from the original ones. Images of cropped blots (top and bottom) are aligned by the same loading control of the purified His-MSMEG_2624 protein (not shown), whereas those of cropped gels (middle) are aligned by the protein ladder (not shown).

Figure 4.

The efficiency of in vivo ribosomal biogenesis of MSMEG_2624 mutants. A, sucrose gradient centrifugation of the WT M. smegmatis, the MSMEG_2624 knockout strain, the MSMEG_2624 knockout with the vector that expresses MSMEG_2624, and the MSMEG_2624 knockout that carries the empty vector. The vertical yellow bar marks the fraction further characterized using MS in B. B, The bar plot shows the ratio of S12/S4 in the 30S subunit of WT, the MSMEG_2624 deletion mutant strain, and the MSMEG_2624 knock-out strain with the vector that expresses MSMEG_2624. C, sucrose gradient centrifugation shows the ribosome profile of the MSMEG_2624 mutants which affects binding with RpsL (ΔP90-D93, shown in green), and which does not (P95G, shown in red). The controls of empty vector and empty vector + WT are shown in cyan and purple.