Volume 24, Issue 10, Pages 1788-1794 (October 2016) Mycobacterium tuberculosis LppM Displays an Original Structure and Domain Composition Linked to a Dual Localization  Philippe Barthe, Romain Veyron-Churlet, Angélique de Visch, Martine Gilleron, Jean-Michel Saliou, Stanislas Tomavo, Jérôme Nigou, Priscille Brodin, Martin Cohen-Gonsaud  Structure  Volume 24, Issue 10, Pages 1788-1794 (October 2016) DOI: 10.1016/j.str.2016.07.009 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 LppM Sequence Analysis (A) LppM hydrophobic cluster analysis (HCA). The secondary structure determined by our NMR analysis is represented on the top (HCA depiction was produced using a web server at http://mobyle.rpbs.univ-paris-diderot.fr/). Symbols are used to represent amino acids with peculiar structural properties: star for proline, black diamond for glycine, square and dotted square for threonine and serine, respectively. The hydrophobic residues are represented in green while the charged residues are represented in red; as expected the lysine and arginine residues are represented in blue. (B) Schematic representation of the different forms of LppM with their relative molecular weights used in this study. The “full-length” LppM corresponds to the entire protein containing a signal peptide (PS) and a transmembrane domain (TM). Structure 2016 24, 1788-1794DOI: (10.1016/j.str.2016.07.009) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 LppM Localization (A) Western blot with pellet (P) and supernatant (S) from both Mtb H37Rv wild-type (WT) and ΔlppM mutant cultures using anti-LppM polyclonal antibodies and anti-Hsp65 monoclonal antibodies, used as a control for the absence of bacterial lysis in the supernatant fraction. (B) Western blot using anti-LppM antibodies of Mtb H37Rv cell fractionation (CW, cell wall; MB, membrane; CYT, cytosol). (C) Anti-LppM western blot using pellet and supernatant of M. smegmatis mc2155 overexpressing lppM. (D) Anti-LppM western blot using a pellet of M. smegmatis mc2155 empty vector pVV16 or overexpressing WT lppM, lppM_C25A and lppM_C25S. Anti-Hsp65 western blot was used as a control for the absence of bacterial lysis in the supernatant fraction. Structure 2016 24, 1788-1794DOI: (10.1016/j.str.2016.07.009) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 LppM Structure (A) [1H,15N]-HSQC spectrum of LppM26-185. This spectrum was obtained at 700 MHz, 20°C, and pH 6.8 with 0.5 mM 15N-uniformly labeled sample. Cross-peak assignments are indicated using the one-letter amino acid code and number following the full-length protein sequence numbering. (B) Cartoon representation of the LppM26-185 structure with the secondary structure numbering. The unique α helix is represented in red while the “degenerated” β strand is represented in orange. (C and D) Two alternative views of the LppM26-185 structure to illustrate and emphasize the lack of hydrophobic cavity within the protein. Structure 2016 24, 1788-1794DOI: (10.1016/j.str.2016.07.009) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Negative MALDI-TOF MS analysis of lipids associated with the purified 6His-LppM26-185 As a negative control, a lysate of M. smegmatis mc2155 containing empty vector (pMV261) was used, following exactly the same steps of concentration and purification. Structure 2016 24, 1788-1794DOI: (10.1016/j.str.2016.07.009) Copyright © 2016 Elsevier Ltd Terms and Conditions