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Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13

Nature Structural & Molecular Biology volume 30, pages 296–308 (2023)Cite this article

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Abstract

The mycolic acid layer of the Mycobacterium tuberculosis cell wall is essential for viability and virulence, and the enzymes responsible for its synthesis are targets for antimycobacterial drug development. Polyketide synthase 13 (Pks13) is a module encoding several enzymatic and transport functions that carries out the condensation of two different long-chain fatty acids to produce mycolic acids. We determined structures by cryogenic-electron microscopy of dimeric multi-enzyme Pks13 purified from mycobacteria under normal growth conditions, captured with native substrates. Structures define the ketosynthase (KS), linker and acyl transferase (AT) domains at 1.8 Å resolution and two alternative locations of the N-terminal acyl carrier protein. These structures suggest intermediate states on the pathway for substrate delivery to the KS domain. Other domains, visible at lower resolution, are flexible relative to the KS-AT core. The chemical structures of three bound endogenous long-chain fatty acid substrates were determined by electrospray ionization mass spectrometry.

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Fig. 1: Overall architecture of the mycobacterial Pks13 showing two alternative positions of the ACP1 domain.
Fig. 2: Single-particle analysis processing workflow from videos through consensus C2 refinement and focused refinements.
Fig. 3: Map-to-model FSCs, and example EM densities.
Fig. 4: Meromycolyl substrate in the KS active site.
Fig. 5: Substrate-bound AT active site, and alternative inward and outward positions of the AT domain.
Fig. 6: C-terminal domains are visualized at low resolution in two alternative positions.
Fig. 7: Proposed pathway to the structures determined for substrates bound Pks13.

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Data availability

The atomic coordinates for five structures of Pks13 have been deposited in the PDB with the accession codes 7UK4, 8CUY, 8CV1, 8CUZ and 8CV0. The corresponding maps have been deposited in the Electron Microscopy Data Bank with the accession codes EMD-26574, EMD-27002, EMD-27005, EMD-27003 and EMD-27004. Proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD033471. Files containing the input data, scripts and output results for integrative modeling are freely available at https://github.com/integrativemodeling/Pks13. The details about the approach are given in Supplementary Table 4. Source data are provided with this paper.

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Acknowledgements

We thank J. Sacchettini, Texas A&M University for the inhibitor TAM16 and C. Sassetti, University of Massachusetts Worcester for the Pks13-TEV-eGFP MSMEG strain and both for their insights, A. Keatinge-Clay and J. Sacchettini for their reading of the paper, counsel and advice, and G. Ashley, ProLynx, Inc. for Supplementary Fig. 1b. We acknowledge P. Thomas, M. Harrington, J. Baker-LePain and the Wynton HPC for computational support; and D. Bulkley, Z. Yu and G. Gilbert for their maintenance of the UCSF EM Core. We thank J. Wilkins and K. Li for performing initial mass spectrometry for lipid identification in the UCSF mass spectrometry core. Research was financially supported by grant nos. P01 AI095208 (J. Sacchettini), GM024485 (R.M.S.), AI48366 (Z.G.), GM083960 and GM109824 (A.S.), U19 A1135990 (N.J.K.) and R01 AI128214 (O.S.R.). M.S.D. acknowledges a National Science Federation graduate fellowship.

Author information

Author notes

  1. These authors contributed equally: Sun Kyung Kim, Miles Sasha Dickinson.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA

    Sun Kyung Kim, Miles Sasha Dickinson, Janet Finer-Moore, Jen Chen, Oren S. Rosenberg & Robert M. Stroud

  2. Chemistry and Chemical Biology Graduate Program, University of California San Francisco, San Francisco, CA, USA

    Miles Sasha Dickinson & Robert M. Stroud

  3. Department of Biochemistry, Duke University Medical Center, Durham, NC, USA

    Ziqiang Guan

  4. Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA, USA

    Robyn M. Kaake, Ernst H. Pulido & Nevan J. Krogan

  5. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

    Robyn M. Kaake, Ignacia Echeverria & Nevan J. Krogan

  6. Quantitative Biosciences Institute, University of California San Francisco, San Francisco, CA, USA

    Robyn M. Kaake, Ignacia Echeverria & Nevan J. Krogan

  7. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA

    Andrej Sali

  8. Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, CA, USA

    Oren S. Rosenberg

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Contributions

O.S.R., S.K.K. and R.M.S. conceived the project. S.K.K. and J.C. prepared the protein. M.S.D. prepared EM grids and performed EM imaging and image processing. M.S.D. interpreted density maps and determined the five structures reported. S.K.K., M.S.D. and J.F.-M. refined and interpreted the structures. Z.G. determined the lipid chains attached to PKS13 by mass spectrometry at ‘the lipidomics project’. E.H.P., R.M.K. and N.J.K. determined the DSSO cross-links formed by mass spectrometry. I.E. and A.S. performed integrative structure determination. R.M.S. and O.S.R. supervised the project. S.K.K., M.S.D., J.F.M., Z.G., O.S.R. and R.M.S. wrote the paper with contributions from all authors.

Corresponding authors

Correspondence to Oren S. Rosenberg or Robert M. Stroud.

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Competing interests

The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche and Rezo Therapeutics. N.J.K. has financially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, Maze Therapeutics, Interline Therapeutics, Rezo Therapeutics, GEn1E Lifesciences, Inc. and Twist Bioscience Corp. He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics and Interline Therapeutics.

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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Florian Ullrich and Carolina Perdigoto, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Purification of native Pks13 from mycobacteria.

(a) Final Superose 6 size exclusion chromatography profile of Pks13. Two peaks containing Pks13 are labeled 1 and 2, in addition to the void peak and peak containing TEV protease. Pks13 peaks were run on non-reducing SDS-PAGE (Biorad #4561096) (b) and blue native PAGE (Invitrogen #BN1004BOX) (c), which were repeated twice with similar results. In SDS-PAGE (b) the protein runs at ~198 kDa, as expected for a protomer of Pks13. Blue native PAGE (c) indicates that majority of the protein is in a dimeric form, and higher oligomeric forms are present. Upon screening both peaks 1 and 2 on gold Quantifoil grids by cryoEM, peak 1 was selected for data collection since it produced more monodisperse particles that correspond to Pks13 dimers. We conclude that the Pks13 dimers associate loosely in solution, and that these readily dissociate to form monodisperse Pks13 dimers under the conditions in which the images were acquired. (d) 78.1% sequence coverage (highlighted red) by tryptic digested Pks13 fragments run on LC-MS-MS.

Source data

Extended Data Fig. 2 DE-rich linker.

17 amino acid linking sequence (E76-92) between ACP1 and KS includes nine acidic amino acids (colored orange) and termed the ‘DE-rich linker’. First 11 amino acids (E76-E86) lie near positively charged surfaces of two different regions on the KS monomer surface (Extended Data Fig. 3). The last six amino acids (D87-R92) closely overlap in orientation between the ACP1a and ACP1b structures and interact electrostatically with the shared positively charged surface (Extended Data Fig. 3).

Extended Data Fig. 3 Electrostatic surface rendering of ACP1-KS-AT domains.

(a) The KS-AT surface is colored by electrostatic potential (red: acidic, blue: basic) of the residues. ACP1a and ACP1b and their ensuing linkers to the KS (yellow and lemon chiffon) are superimposed onto the KS-AT electrostatic potential surface, illustrating that the DE-rich linker lie near positively charged surfaces on the KS. (b) ACP1a, ACP1b, and the linkers to the KS are colored by electrostatic potential (red: acidic, blue: basic) of the residues. Model of KS-AT is superimposed onto the highly negative electrostatic potential surface of the DE-rich linker.

Extended Data Fig. 4 Interfacing residues between DE-rich linker and KS domain in ACP1a position.

Interfacing residues were determined by PISA and labeled. Hydrogen bond between E88(OE2):R384(NH2) is represented with a yellow dotted line.

Extended Data Fig. 5 Interfacing residues between DE-rich linker and KS domain in ACP1b position.

Interfacing residues were determined by PISA and labeled. Hydrogen bonds between Y84(OH):R395(NH1), E88(OE2):R384(NH1), and E88(OE2):K388(NZ) are labeled with yellow dotted lines.

Extended Data Fig. 6 ACP1a interaction with KS, KS-AT linker domain, and AT-ACP2 linker.

Interfacing residues between ACP1a and KS, KS-AT linker domain, and AT-ACP2 linker are shown as stick representation and labeled. Pi-pi stacking interaction is shown between R63 of ACP1a and R496 of KS. The Ppant arm is shown attached to S38 in helix II of ACP1a, and Ppant density is shown zoned around 2.3 Å around the model.

Extended Data Fig. 7 ACP1b interaction with KS and AT’.

(a) ACP1b:AT’ interfacing residues’ side chains are shown and labeled, revealing a hydrophilic interface. (b) ACP1b:KS interfacing residues’ side chains are shown and labeled, revealing a hydrophobic interface.

Extended Data Fig. 8 The active center of the AT domain shows the bound substrate interactions.

(a) Crossed-eyes stereo figure showing the density for the active site of the AT domain with substrate bound. The α-carboxyl group on the bound substrate is stabilized by a π-anion interaction with the phenyl ring of F709. (b) Rotated ∼90° around the vertical axis and tilted forward from panel (a) shows interactions between the active center of the AT domain and the bound α-carboxy acyl substrate chain attached to S798. Distances between heavy atoms are tabulated in Å. The catalytic base H906 is ideally placed for the reaction at the γO of S798. The ester carbonyl on the substrate, and hence the oxyanion intermediate formed at that oxygen during the catalytic reaction may be stabilized by R823, H906, and S905. The H906 orientation is aligned by hydrogen bonds from the δN-H to the C = O of G959 and of H962.

Extended Data Fig. 9 Linkage map depicting all unique K-K linkages of DSSO cross-linked Pks13.

The 57 unique K-K inter-linked residues are summarized in this linkage map as black lines connecting two Pks13 lysine residues. Shown is a cartoon diagram of the primary sequence structure of Pks13 with domains labeled as in Fig. 1a. The sequence numbers of lysine residues indicated by linear position in this figure are included in Supplementary Tables 2, 3, 5, and 6.

Extended Data Fig. 10 Integrative structure modeling of the full-length Pks13 dimer.

(a) The localization probability density of the ensemble of structures is shown with representative (centroid) structure from the ensemble embedded within it. The three panels represent different solutions with their relative percentages listed above. The structured and unstructured regions are represented as beads, using the same color scheme as Fig. 1a. Regions with well-defined structures are modeled as rigid bodies consisting of beads representing each individual residue; the coordinates of a 1-residue bead were those of the corresponding Cα atom. Regions with no available structure or flexible regions are represented as flexible strings of beads, corresponding to up to 10 residues per bead. (b) Detail of cross-links mapped to the centroid structure of the integrative model of the Pks13 dimer. Satisfied and violated cross-links shown in blue and yellow, respectively. A cross-link is classified as satisfied if the Cα–Cα distance spanned by the cross-linked residues in any of the models of the cluster is less than 35 Å. (c) Histogram showing the distribution of the cross-linked Cα–Cα distances in the Pks13 dimer integrative structures. (d) Histogram showing the Cα–Cα distances between the active sites of the ACP1-KS, AT-ACP2, KS-ACP2, and ACP2-TE domains. The shortest distances (red lines) are 26, 13.7, 14.8, and 9.7 Å between the ACP1-KS, AT-ACP2, KS-ACP2, and ACP2-TE domains, respectively.

Supplementary information

Supplementary Information

Supplementary Text, Figs. 1–13, Table 1, captions for Tables 2–6, caption for Video 1 and References.

Reporting Summary

Supplementary Table 2

Summary of unique Pks13 interlinked residues excluding files from highly cross-linked samples.

Supplementary Table 3

Metadata of all DSSO cross-linked Pks13 XL–MS files.

Supplementary Table 5

Table with description and scores for overall DSSO cross-linked peptides for all processed files including interlinked, monolinked and single peptides.

Supplementary Table 6

Summary of all unique Pks13 interlinked residues from the full dataset.

Supplementary Video 1

Relative positions of the ACP1, KS, AT domains and substrates determined by cryo-EM.

Source data

Source Data Fig. 1

Chem draw data for Fig. 1 trehalose.

Source Data Fig. 1

Chem draw data for Fig. 1 mechanism.

Source Data Extended Data Fig. 1

Uncropped gel pictures.

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Kim, S.K., Dickinson, M.S., Finer-Moore, J. et al. Structure and dynamics of the essential endogenous mycobacterial polyketide synthase Pks13. Nat Struct Mol Biol 30, 296–308 (2023). https://doi.org/10.1038/s41594-022-00918-0

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