Skip to main content
SpringerLink
Log in

The Structural Features of MlaD Illuminate its Unique Ligand-Transporting Mechanism and Ancestry

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

The membrane-associated solute-binding protein (SBP) MlaD of the maintenance of lipid asymmetry (Mla) system has been reported to help the transport of phospholipids (PLs) between the outer and inner membranes of Gram-negative bacteria. Despite the availability of structural information, the molecular mechanism underlying the transport of PLs and the ancestry of the protein MlaD remain unclear. In this study, we report the crystal structures of the periplasmic region of MlaD from Escherichia coli (EcMlaD) at a resolution range of 2.3–3.2 Å. The EcMlaD protomer consists of two distinct regions, viz. N-terminal β-barrel fold consisting of seven strands (referred to as MlaD domain) and C-terminal α-helical domain (HD). The protein EcMlaD oligomerizes to give rise to a homo-hexameric ring with a central channel that is hydrophobic and continuous with a variable diameter. Interestingly, the structural analysis revealed that the HD, instead of the MlaD domain, plays a critical role in determining the oligomeric state of the protein. Based on the analysis of available structural information, we propose a working mechanism of PL transport, viz. “asymmetric protomer movement (APM)”. Wherein half of the EcMlaD hexamer would rise in the periplasmic side along with an outward movement of pore loops, resulting in the change of the central channel geometry. Furthermore, this study highlights that, unlike typical SBPs, EcMlaD possesses a fold similar to EF/AMT-type beta(6)-barrel and a unique ancestry. Altogether, the findings firmly establish EcMlaD to be a non-canonical SBP with a unique ligand-transport mechanism.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data Availability

All the data are submitted along with the manuscript. The three-dimensional atomic coordinates and the structure factors have been deposited in the RCSB Protein Data Bank with the accession codes 8HQA, 8HPZ and 8HQ9.

Abbreviations

ABC:

ATP-binding cassette

ATP:

adenosine triphosphate

APM:

asymmetric protomer movement

CTD:

C-terminal domain

IF:

interfacial

IM:

inner membrane

LPS:

lipopolysaccharide

LTP:

lipid-transfer proteins

Mla:

maintenance of lipid asymmetry

NTD:

N-terminal domain

OM:

outer membrane

PDB:

protein data bank

PEF:

phosphatidylethanolamine

PL:

phospholipid

PLP:

pore loop

RMSD:

root-mean-square deviation

SBP:

solute-binding protein

SDM:

segmented domain movement

TMD:

transmembrane domain

References

  1. Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Delcour AH (2009) Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta - Proteins Proteom 1794:808–816

    Article  CAS  Google Scholar 

  3. Henderson JC, Zimmerman SM, Crofts AA, Boll JM, Kuhns LG, Herrera CM, Trent MS (2016) The power of asymmetry: architecture and assembly of the Gram-negative outer membrane lipid bilayer. Annu Rev Microbiol 70:255–278

    Article  CAS  PubMed  Google Scholar 

  4. Bishop RE (2008) Structural biology of membrane-intrinsic β-barrel enzymes: sentinels of the bacterial outer membrane. Biochim Biophys Acta - Biomembr 1778:1881–1896

    Article  CAS  Google Scholar 

  5. Malinverni JC, Silhavy TJ (2009) An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane. Proc Natl Acad Sci USA 106:8009–8014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hughes GW, Hall SC, Laxton CS, Sridhar P, Mahadi AH, Hatton C, Piggot TJ, Wotherspoon PJ, Leney AC, Ward DG, Jamshad M, Spana V, Cadby IT, Harding C, Isom GL, Bryant JA, Parr RJ, Yakub Y, Jeeves M, Huber D, Henderson IR, Clifton LA, Lovering AL, Knowles TJ (2019) Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system. Nat Microbiol 4:1692–1705

    Article  CAS  PubMed  Google Scholar 

  7. Coudray N, Isom GL, MacRae MR, Saiduddin MN, Bhabha G, Ekiert DC (2020) Structure of bacterial phospholipid transporter MlaFEDB with substrate bound. eLife 9:e62518

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Low WY, Thong S, Chng SS (2021) ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry. Proc Natl Acad Sci USA 118:e2110055118

    Article  PubMed  PubMed Central  Google Scholar 

  9. Ekiert DC, Coudray N, Bhabha G (2022) Structure and mechanism of the bacterial lipid ABC transporter, MlaFEDB. Curr Opin Struct Biol 76:102429

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rees DC, Johnson E, Lewinson O (2009) ABC transporters: the power to change. Nat Rev Mol Cell Biol 10:218–227

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wilkens S (2015) Structure and mechanism of ABC transporters. F1000Prime Rep 7:14

    Article  PubMed  PubMed Central  Google Scholar 

  12. Maqbool A, Horler RS, Muller A, Wilkinson AJ, Wilson KS, Thomas GH (2015) The substrate-binding protein in bacterial ABC transporters: dissecting roles in the evolution of substrate specificity. Biochem Soc Trans 43:1011–1017

    Article  CAS  PubMed  Google Scholar 

  13. Berntsson RP, Smits SH, Schmitt L, Slotboom DJ, Poolman B (2010) A structural classification of substrate-binding proteins. FEBS Lett 584:2606–2617

    Article  CAS  PubMed  Google Scholar 

  14. Fukami-Kobayashi K, Tateno Y, Nishikawa K (1999) Domain dislocation: a change of core structure in periplasmic binding proteins in their evolutionary history. J Mol Biol 286:279–290

    Article  CAS  PubMed  Google Scholar 

  15. Lee YH, Deka RK, Norgard MV, Radolf JD, Hasemann CA (1999) Treponema pallidum TroA is a periplasmic zinc-binding protein with a helical backbone. Nat Struct Mol Biol 6:628–633

    Article  CAS  Google Scholar 

  16. Scheepers GH, Lycklama a Nijeholt JA, Poolman B (2016) An updated structural classification of substrate-binding proteins. FEBS Lett 590:4393–4401

    Article  CAS  PubMed  Google Scholar 

  17. Chandravanshi M, Tripathi SK, Kanaujia SP (2021) An updated classification and mechanistic insights into ligand binding of the substrate-binding proteins. FEBS Lett 595:2395–2409

    Article  CAS  PubMed  Google Scholar 

  18. Dutta A, Kanaujia SP (2022) MlaC belongs to a unique class of non-canonical substrate-binding proteins and follows a novel phospholipid-binding mechanism. J Struct Biol 214:107896

    Article  CAS  PubMed  Google Scholar 

  19. Wong LH, Gatta AT, Levine TP (2019) Lipid transfer proteins: the lipid commute via shuttles, bridges and tubes. Nat Rev Mol Cell Biol 20:85–101

    Article  CAS  PubMed  Google Scholar 

  20. Arruda S, Bomfim G, Knights R, Huima-Byron T, Riley LW (1993) Cloning of an M. Tuberculosis DNA fragment associated with entry and survival inside cells. Science 261:1454–1457

    Article  CAS  PubMed  Google Scholar 

  21. Ekiert DC, Bhabha G, Isom GL, Greenan G, Ovchinnikov S, Henderson IR, Cox JS, Vale RD (2017) Architectures of lipid transport systems for the bacterial outer membrane. Cell 169:273–285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dutta A, Chandravanshi M, Kanaujia SP (2021) Conserved features of the MlaD domain aid the trafficking of hydrophobic molecules. Proteins 89:1473–1488

    Article  CAS  PubMed  Google Scholar 

  23. Isom GL, Coudray N, MacRae MR, McManus CT, Ekiert DC, Bhabha G (2020) LetB structure reveals a tunnel for lipid transport across the bacterial envelope. Cell 181:653–664

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hassell AM, An G, Bledsoe RK, Bynum JM, Carter HL, Deng SJ, Gampe RT, Grisard TE, Madauss KP, Nolte RT, Rocque WJ (2007) Crystallization of protein-ligand complexes. Acta Crystallogr D Biol Crystallogr 63:72–79

    Article  CAS  PubMed  Google Scholar 

  25. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67:271–281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Evans PR, Murshudov GN (2013) How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr 69:1204–1214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67:235–242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40:658–674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Brünger AT (1992) Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355:472–475

    Article  PubMed  Google Scholar 

  30. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Vagin AA, Steiner RA, Lebedev AA, Potterton L, McNicholas S, Long F, Murshudov GN (2004) REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr D Biol Crystallogr 60:2184–2195

    Article  PubMed  Google Scholar 

  32. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291

    Article  CAS  Google Scholar 

  33. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr D66:12–21

    Article  Google Scholar 

  34. Berman HM, Bhat TN, Bourne PE, Feng Z, Gilliland G, Weissig H, Westbrook J (2000) The protein data bank and the challenge of structural genomics. Nat Struct Mol Biol 7:957–959

    Article  CAS  Google Scholar 

  35. The UniProt Consortium (2022) UniProt: the universal protein knowledgebase in 2023. Nucleic Acids Res 51:D523–D531

    Article  Google Scholar 

  36. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  37. Sievers F, Higgins DG (2014). In: Russell D (ed) Methods in Molecular Biology. Humana Press, Totowa

    Google Scholar 

  38. Gouet P, Robert X, Courcelle E (2003) ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res 31:3320–3323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lomize MA, Pogozheva ID, Joo H, Mosberg HI, Lomize AL (2012) OPM database and PPM web server: resources for positioning of proteins in membranes. Nucleic Acids Res 40:D370–D376

    Article  CAS  PubMed  Google Scholar 

  41. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797

    Article  CAS  PubMed  Google Scholar 

  42. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596:583–589

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Varadi M, Anyango S, Deshpande M, Nair S, Natassia C, Yordanova G, Yuan D, Stroe O, Wood G, Laydon A, Žídek A (2022) AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res 50:D439–D444

    Article  CAS  PubMed  Google Scholar 

  44. Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577–2637

    Article  CAS  PubMed  Google Scholar 

  45. Joosten RP, Te Beek TA, Krieger E, Hekkelman ML, Hooft RW, Schneider R, Sander C, Vriend G (2010) A series of PDB related databases for everyday needs. Nucleic Acids Res 39:D411–D419

    Article  PubMed  PubMed Central  Google Scholar 

  46. Laskowski RA, Jabłońska J, Pravda L, Vařeková RS, Thornton JM (2018) PDBsum: structural summaries of PDB entries. Protein Sci 27:129–134

    Article  CAS  PubMed  Google Scholar 

  47. Bond CS (2003) TopDraw: a sketchpad for protein structure topology cartoons. Bioinformatics 19:311–312

    Article  CAS  PubMed  Google Scholar 

  48. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  CAS  PubMed  Google Scholar 

  49. Chovancova E, Pavelka A, Benes P, Strnad O, Brezovsky J, Kozlikova B, Gora A, Sustr V, Klvana M, Medek P, Biedermannova L (2012) CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput Biol 8:e1002708

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hagemans D, van Belzen IA, Morán Luengo T, Rüdiger SG (2015) A script to highlight hydrophobicity and charge on protein surfaces. Front Mol Biosci 2:56

    Article  PubMed  PubMed Central  Google Scholar 

  51. Holm L (2022) Dali server: structural unification of protein families. Nucleic Acids Res 50:W210–W215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer EL, Tosatto SC, Paladin L, Raj S, Richardson LJ, Finn RD (2021) Pfam: the protein families database in 2021. Nucleic Acids Res 49:D412–D419

    Article  CAS  PubMed  Google Scholar 

  53. Blum M, Chang HY, Chuguransky S, Grego T, Kandasaamy S, Mitchell A, Nuka G, Paysan-Lafosse T, Qureshi M, Raj S, Richardson L, Salazar GA, Williams L, Bork P, Bridge A, Gough J, Haft DH, Letunic I, Marchler-Bauer A, Mi H, Natale DA, Necci M, Orengo CA, Pandurangan AP, Rivoire C, Sigrist CJA, Sillitoe I, Thanki N, Thomas PD, Tosatto SCE, Wu CH, Bateman A, Finn RD (2021) The InterPro protein families and domains database: 20 years on. Nucleic Acids Res 49:D344–D354

    Article  CAS  PubMed  Google Scholar 

  54. Dawson NL, Lewis TE, Das S, Lees JG, Lee D, Ashford P, Orengo CA, Sillitoe I (2017) CATH: an expanded resource to predict protein function through structure and sequence. Nucleic Acids Res 45:D289–D295

    Article  CAS  PubMed  Google Scholar 

  55. Andreeva A, Kulesha E, Gough J, Murzin AG (2020) The SCOP database in 2020: expanded classification of representative family and superfamily domains of known protein structures. Nucleic Acids Res 48:D376–D382

    Article  CAS  PubMed  Google Scholar 

  56. Shukolyukov SA (2009) Aggregation of frog rhodopsin to oligomers and their dissociation to monomer: application of BN-and SDS-PAGE. Biochem (Moscow) 74:599–604

    Article  CAS  Google Scholar 

  57. Asthana P, Singh D, Pedersen JS, Hynönen MJ, Sulu R, Murthy AV, Laitaoja M, Jänis J, Riley LW, Venkatesan R (2021) Structural insights into the substrate-binding proteins Mce1A and Mce4A from Mycobacterium tuberculosis. IUCrJ 8:757–774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Buslaev P, Gordeliy V, Grudinin S, Gushchin I (2016) Principal component analysis of lipid molecule conformational changes in molecular dynamics simulations. J Chem Theory Comput 12(3):1019–1028

    Article  CAS  PubMed  Google Scholar 

  59. Neumann J, Rose-Sperling D, Hellmich UA (2017) Diverse relations between ABC transporters and lipids: an overview. Biochim Biophys Acta - Biomembr 1859:605–618

    Article  CAS  PubMed  Google Scholar 

  60. Casali N, Riley LW (2007) A phylogenomic analysis of the Actinomycetales mce operons. BMC Genom 8(1):1–23

    Article  Google Scholar 

  61. Thong S, Ercan B, Torta F, Fong ZY, Wong HY, Wenk MR, Chng SS (2016) Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry. eLife 5:e19042

    Article  PubMed  PubMed Central  Google Scholar 

  62. Asthana P, Singh D, Pedersen JS, Hynönen MJ, Sulu R, Murthy AV, Laitaoja M, Jänis J, Riley LW, Venkatesan R (2021) Structural insights into the substrate-binding proteins Mce1A and Mce4A from Mycobacterium tuberculosis. IUCrJ 8(5):757–774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Rank L, Herring LE, Braunstein M (2021) Evidence for the mycobacterial Mce4 transporter being a multiprotein complex. J Bacteriol 203(10):10–128

    Article  Google Scholar 

  64. Yero D, Díaz-Lobo M, Costenaro L, Conchillo-Solé O, Mayo A, Ferrer-Navarro M, Vilaseca M, Gibert I, Daura X (2021) The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm. Commun Biol 4(1):448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Schultz KM, Fischer MA, Noey EL, Klug CS (2018) Disruption of the E. Coli LptC dimerization interface and characterization of lipopolysaccharide and LptA binding to monomeric LptC. Protein Sci 27:1407–1417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Sandhya S, Rani SS, Pankaj B, Govind MK, Offmann B, Srinivasan N, Sowdhamini R (2009) Length variations amongst protein domain superfamilies and consequences on structure and function. PLoS ONE 4:e4981

    Article  PubMed  PubMed Central  Google Scholar 

  67. Asada K (2006) Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 141(2):391–396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Mao B, Pear MR, McCammon JA, Quiocho FA (1982) Hinge-bending in L-arabinose-binding protein. The Venus’s-flytrap model. J Biol Chem 257:1131–1133

    Article  CAS  PubMed  Google Scholar 

  69. Pandey S, Modak A, Phale PS, Bhaumik P (2016) High resolution structures of periplasmic glucose-binding protein of Pseudomonas putida CSV86 reveal structural basis of its substrate specificity. J Biol Chem 291:7844–7857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Chandravanshi M, Gogoi P, Kanaujia SP (2020) Structural and thermodynamic correlation illuminates the selective transport mechanism of disaccharide α-glycosides through ABC transporter. FEBS J 287:1576–1597

    Article  CAS  PubMed  Google Scholar 

  71. Chandravanshi M, Samanta R, Kanaujia SP (2020) Conformational trapping of a β-Glucosides-binding protein unveils the selective two-step ligand-binding mechanism of ABC importers. J Mol Biol 432:5711–5734

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The work was supported by the Science and Engineering Research Board (SERB), Department of Science and Technology, Government of India (Grant number: ECR/2018/000013). The authors acknowledge the Central Instruments Facility (CIF) at the Indian Institute of Technology Guwahati (IITG) for providing the X-ray diffractometer (XRD). The authors are grateful to all the members of the Structural and Computational Biology laboratory (SCBL) for their continuous support. AD acknowledges the Ministry of Human Resource and Development (MHRD), Government of India, for providing the research fellowship.

Author information

Authors and Affiliations

  1. Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Angshu Dutta & Shankar Prasad Kanaujia

Authors
  1. Angshu Dutta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shankar Prasad Kanaujia
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SPK: conceived the project, designed the study, and solved the structures; AD: performed the experiments; SPK and AD: analyzed and validated the data; SPK and AD: wrote the manuscript.

Corresponding author

Correspondence to Shankar Prasad Kanaujia.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dutta, A., Kanaujia, S.P. The Structural Features of MlaD Illuminate its Unique Ligand-Transporting Mechanism and Ancestry. Protein J (2024). https://doi.org/10.1007/s10930-023-10179-5

Download citation

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10930-023-10179-5

Keywords

Search

Navigation