Identification of specific posttranslational O-mycoloylations mediating protein targeting to the mycomembrane

Abstract

The outer membranes (OMs) of members of the Corynebacteriales bacterial order, also called mycomembranes, harbor mycolic acids and unusual outer membrane proteins (OMPs), including those with α-helical structure. The signals that allow precursors of such proteins to be targeted to the mycomembrane remain uncharacterized. We report here the molecular features responsible for OMP targeting to the mycomembrane of Corynebacterium glutamicum, a nonpathogenic member of the Corynebacteriales order. To better understand the mechanisms by which OMP precursors were sorted in C. glutamicum, we first investigated the partitioning of endogenous and recombinant PorA, PorH, PorB, and PorC between bacterial compartments and showed that they were both imported into the mycomembrane and secreted into the extracellular medium. A detailed investigation of cell extracts and purified proteins by top-down MS, NMR spectroscopy, and site-directed mutagenesis revealed specific and well-conserved posttranslational modifications (PTMs), including O-mycoloylation, pyroglutamylation, and N-formylation, for mycomembrane-associated and -secreted OMPs. PTM site sequence analysis from C. glutamicum OMP and other O-acylated proteins in bacteria and eukaryotes revealed specific patterns. Furthermore, we found that such modifications were essential for targeting to the mycomembrane and sufficient for OMP assembly into mycolic acid-containing lipid bilayers. Collectively, it seems that these PTMs have evolved in the Corynebacteriales order and beyond to guide membrane proteins toward a specific cell compartment.

Keywords: Corynebacteriales; NMR; O-acylation; sequence motif; top-down proteomics.

Fig. 1.

Identification of distinct proteoforms for C. glutamicum OMPs associated with the mAGP complex and secreted in the extracellular medium. (A) C. glutamicum ATCC13032 cells expressing recombinant PorA-His, PorH-His, PorB-His, and PorC-His were cultured under identical conditions. Subcellular fractions corresponding to the CYT, the PM, the mAGP complex, and the extracellular medium were analyzed by SDS/PAGE after staining with InstantBlue (Left) and Western blotting (Right) with antibodies against the protein His tag. Fractions isolated from WT, untransformed cells were coanalyzed as control. Molecular mass markers (in kilodaltons) are indicated next to the gel. (B) Extracted ion chromatograms of PorB-His purified from mAGP (Upper) and extracellular medium (Lower) fractions containing nonmycoloylated (black), monomycoloylated (green), and dimycoloylated (red) proteoforms. (C) Representation of the multicharged MS spectra (Left) and deconvoluted (DC) spectra obtained for PorB-His proteoforms with isotopic resolution (Right). The mycolic acid compositions of each proteoform are indicated by triangle and circle symbols. EM, extracellular medium.

Fig. 2.

Characterization of protein modifications by MS/MS, NMR, and site-directed mutagenesis. Example of PorB. (A) Top-down CID of dimycoloylated PorB-His-10+ charge state (m/z 1,336.10 Th) with nonmycoloylated, monomycoloylated, and dimycoloylated y and b fragments colored black, green, and red, respectively. The sequence coverage was obtained by fragmenting the 8+, 9+, and 10+ charge states of PorB-His, identifying S98 and S7/S8 residues (polygons) as putative mycoloylation sites and a disulfide bond between C22 and C81 (yellow line). (B) Solution NMR analysis of nonmycoloylated (black) and mycoloylated (red) PorB-His. Selected strips extracted from 3D ¹H, ¹⁵N, ¹H heteronuclear single quantum coherence–total correlation spectroscopy (HSQC-TOCSY) spectra obtained on (U-¹⁵N)–labeled PorB-His showing Hα and Hβ₂ chemical shifts of S7 and S8 residues for the two proteoforms. Although ¹H resonances from S8 were not affected, significant spectral changes were observed for Hα of residue S7 (blue arrows), thus identifying the O-acylation of the S7 hydroxyl of PorB-His. (C) The positions of PTM within the protein sequence were validated by site-directed mutagenesis of S7 and S98 residues and subsequent MS analysis of PorB-His WT (WT-mAGP; Top) and its mutant derivatives PorB-S98A (S98A-mAGP; Middle) and PorB-S7AS98A (S7AS98A-mAGP; Bottom). Nonmycoloylated (black), monomycoloylated (green), and dimycoloylated (red) proteoforms were semiquantified from extracted ion chromatograms of the corresponding 9+ charge states.

Fig. S1.

Nano–LC-ESI-MS analysis of recombinant PorA, PorH, and PorC proteoforms purified from extracellular medium (EM) and mAGP fractions. The multicharged MS spectra are shown in Left together with their corresponding isotopic patterns deconvoluted with MagTran. The charge states are indicated in red. C32:0/C34:1/C36:2, mycolic acid composition; ESI, electrospray ionization; fM1, formylation of N-terminal methionine; pE1, N-terminal pyroglutamylation; pS51, phosphorylation of S51 residue; SS, disulfide bridge.

Fig. S2.

Nonacylated OMP isoforms revealed from crude extracellular medium extracts of C. glutamicum using top-down proteomics in discovery mode. (A) Proteoform footprint of the LC-MS run generated with the RoWinPro program, showing the deconvoluted molecular masses obtained along the gradient for extracellular medium extracts of untransformed C. glutamicum WT (red) and ΔcMytC mutant (black). The size of the dots reflects the relative signal intensity for each one of the detected proteins. PorB, PorH, and PorA are indicated with arrows. (B) MS/MS CID spectra were then automatically searched against the C. glutamicum protein database using ProteomeDiscoverer (ThermoFisher Scientific) with the dedicated ProSight-PD nodes. N-formylmethionine is indicated by pink squares, and disulfide bonds are indicated by gray squares. PCS, protein characterization score.

Fig. S3.

MS/MS fragmentation and product ion maps from CID spectra for mono- and multiacylated PorB-His and PorC-His species. (A) Full MS/MS spectrum of monomycoloylated PorB-His (proteoforms 12 and 15; C34:1) 10+ charge state at m/z 1,285.66 Th showing mycoloylation on either S98 or S7 residues (polygons). (B) Low and high m/z ranges of the MS/MS spectrum of the monomycoloylated PorC-His (proteoform 24; C34:1) 9+ charge state (m/z 1,414.40) indicating a pyroglutamylation at position 1, a disulfide bond between C12 and C69, and a mycolic acid on S91 (polygon). The sequence coverage was obtained by fragmentation of the charge states 8+ to 11+. (C) Low and high m/z ranges of the MS/MS spectrum of dimycoloylated PorC-His (proteoform 28; 2xC34:1) 9+ charge state at m/z 1,470.57 Th showing a pyroglutamylation at position 1, a disulfide bond between C12 and C69, and mycolic acids on S91 and S87 residues (polygons). The sequence coverage was obtained by fragmentation of the charge states 8+, 9+, and 10+. The color code used for fragmentation ions y and b is unmodified fragments (black), disulfide bond (−2 Da; yellow), pyroglutamylation (−17 Da; light pink), monomycoloylation (dark green), monomycoloylation and pyroglutamylation (light green), and dimycoloylation (red). Mycoloylations are associated with mass discrepancies of +478 Da (C32:0), +504 Da (C34:1), and +530 Da (C36:2).

Fig. S4.

NMR assignments of backbone and sidechain ¹H and ¹⁵N resonances within the PorB L6-N10 protein segment. (A) 2D (15N-1H)-band-selective optimized-flip-angle short-transient (SOFAST) heteronuclear multiple quantum correlation spectroscopy (HMQC) spectrum obtained on nonacylated (U-¹⁵N)–labeled PorB (black). (B) Sequential strips extracted from the 3D (¹⁵N, ¹H, ¹H)-HSQC-TOCSY spectrum (80 ms mixing time) of nonacylated (U-¹⁵N)–labeled PorB at ¹⁵N and ¹H amide frequencies indicated at the top and the bottom of the strips, respectively. Interresidue correlations between backbone amide resonances of residue i and sidechain proton resonances of residue i-1 are observed, and the corresponding assignments are indicated.

Fig. S5.

MS and MS/MS analysis of PorB-His and PorC-His mutants by using top-down CID. (A, Upper) Multicharged MS spectra of secreted PorB WT (PorB-WT, proteoform 10), single (PorB-S98A) and double (PorB-S7AS98A) mutants, and the corresponding isotopic patterns deconvoluted with MagTran. The charge states are indicated in red. (A, Lower) Full MS/MS spectrum of mAGP-associated PorB-S98A 10+ charge state (m/z 1,284.05) showing the PTM loss in the C-terminal region and the remaining mycoloylation in the S7-L13 protein stretch. The sequence coverage was obtained by fragmentation of the charge states 8+, 9+, and 10+. (B, Upper) Multicharged MS spectra of secreted PorC-His WT (PorC-WT, proteoform 22), single (PorC-S91A) and double (PorC-S87AS91A) mutants, and the corresponding isotopic patterns deconvoluted with MagTran software. The charge states are indicated in red. (B, Lower) Full MS/MS spectrum of mAGP-associated PorC-S91A 10+ charge state (m/z 1,271.46) indicating a unique mycoloylation on S87 residue, thus confirming the PTM loss in the most distal position. The sequence coverage was obtained by fragmentation of the charge states 8+, 9+, and 10+. The color code used for fragmentation ions y and b is unmodified fragments (black), disulfide bond (−2 Da; yellow), pyroglutamylation (−17 Da; light pink), monomycoloylation (dark green), and monomycoloylation and pyroglutamylation (light green). Mycoloylations are associated with mass discrepancies of +478 Da (C32:0), +504 Da (C34:1), and +530 Da (C36:2). The mutated residue is colored in red in the protein sequence.

Fig. 3.

O-acylation of OMPs occurs in short linear motifs. (A) Schematic representation of protein modifications along the protein sequences of C. glutamicum PorA, PorH, PorB, and PorC. The sites of modifications are reported onto the sequence using the following code: M, O-mycoloylation; pE, pyroglutamate; and S-S, disulfide bond. (B) Sequence alignments of selected O-acylated proteins from C. glutamicum and human genomes within a region surrounding the fatty acylation residue. The sequence logo was calculated from the C. glutamicum peptide sequences only and displays the position-specific frequency of each amino acid composing the motif. AA, amino acid; CG, C. glutamicum; HS, Homo sapiens. *Fatty acylation residue.

Fig. S6.

Multiple sequence alignments identify O-acylation short linear motifs within porin orthologs. Multiple sequence alignments for PorA, PorH, PorB, and PorC proteins. O-acylation motifs are underlined, and PTM sites are indicated by arrows. As a reference, the query protein sequences from C. glutamicum ATCC13032 are indicated in the first line, with residue numbers at the top of the alignment. Sequence similarity is shown by red letters, whereas sequence identity is highlighted by white letters on a red background. Orthologous sequences are represented with their respective GenBank accession numbers, and corresponding species are indicated as follows: C. afe (Corynebacterium afermentas); C. cal (Corynebacterium callunae); C. cam (Corynebacterium camporealensis); C. cap (Corynebacterium capitovis); C. cas (Corynebacterium casei); C. des (Corynebacterium deserti); C. dip (Corynebacterium diphtheriae); C. doo (Corynebacterium doosanense); C. eff (Corynebacterium efficiens); C. glu (C. glutamicum); C. hal (Corynebacterium halotolerans); C. hum (Corynebacterium humireducens); C. imi (Corynebacterium imitans); C. lip (Corynebacterium lipophiloflavum); C. lub (Corynebacterium lubricantis); C. mar (Corynebacterium maris); C. pilosum (Corynebacterium pilosum); C. pse (Corynebacterium pseudotuberculosis); C. rie (Corynebacterium riegelii); C. sim (Corynebacterium simulans); C. sta (Corynebacterium stationis); C. str (Corynebacterium striatum); C. tes (Corynebacterium testudinoris); C. tim (Corynebacterium timonense); C. ulc (Corynebacterium ulcerans).

Fig. 4.

The O-acylation is essential for retention of PorH, PorB, and PorC at the mycomembrane but not PorA. (A) The mAGP complex and the PM were isolated from WT (W) and cMytC-deficient (Δ) C. glutamicum strains expressing recombinant PorA-His, PorH-His, PorB-His, and PorC-His and analyzed by SDS/PAGE after immunodecoration with antibodies against protein His tag. (B) Analysis of the mAGP complex purified from WT C. glutamicum cells expressing native recombinant proteins (WT) or the mutant derivatives of PorB-His [i.e., PorB-S7A, PorB-S98A, and PorB-S7AS98A (Left)] and PorC-His [i.e., PorC-S87A, PorC-S91A, and PorC-S87AS91A (Right)] by SDS/PAGE and immunodecoration with antibodies against protein His tag. Fractions isolated from untransformed WT cells were coanalyzed as control. Bands corresponding to the recombinant proteoforms are indicated with arrows using the following code: M, mature form corresponding to the recombinant protein without its N-terminal signal sequence; M*, mature form without O-mycoloylation; P, recombinant protein precursor containing the N-terminal signal sequence. Molecular mass (MW) markers (in kilodaltons) are indicated next to the gel.

Fig. S7.

Characterization of aggregated recombinant PorC-His precursor within mAGP and PM fractions. (A) SDS/PAGE analysis of PorC-His containing C. glutamicum CE fractions before and after treatment with 8 M urea buffer and ultracentrifugation (200,000 × g for 1 h at 4 °C). M, mature form corresponding to the recombinant protein without its N-terminal signal sequence; P, recombinant protein precursor containing the N-terminal signal sequence. (B) Multicharged MS spectrum (Upper) and the corresponding isotopic pattern (Lower) of PorC-His found in the 8 M urea soluble fraction deconvoluted with MagTran software. The charge states are indicated in red. (C) Protein sequence of PorC-His precursor with the N-terminal sequence (italics). The molecular mass is expected to be 14,809.1 Da. The deconvoluted spectrum shows one major species at 14,807.5 Da, which corresponds to the nonmycoloylated PorC-His precursor containing a disulfide bond.

Fig. 5.

Model for the biogenesis of major OMPs in C. glutamicum. After synthesis in the CYT and translocation across the PM through Sec-dependent (PorB and PorC) or –independent (PorA and PorH) pathways, OMPs are transported into the perisplam and acylated with one or several mycolic acids by the cMytC on serine residues located within short linear motifs. Specific posttranslational O-mycoloylations of OMPs are essential for targeting to the mycomembrane and may be involved in the subsequent formation of pore-forming assemblies. Nonacylated proteoforms are released in the extracellular medium (EM), but their function remains to be elucidated. AG, arabinogalactan; PG, peptidoglycan; M, O-mycoloylation; MM, mycomembrane; PM, plasma membrane; SEC, general secretion pathway; TAT, twin arginine translocation pathway.