Molecular and Structural Mechanism for Beta Barrel Proteins Incorporation in Cells
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Beta Barrel Proteins are important for membrane processes. This presentation is a simplified explanation of research article which elaborate incorporation of beta barrel proteins transport and incorporation and secretion snapshot from outer bacterial cell wall.
Molecular and Structural Mechanism for Beta Barrel Proteins Incorporation in Cells
- 1. Biogenesis of 硫-barrel Membrane Proteins Simplified elaboration of beta barrel protein incorporation mechanisms in cells as cited under: Noinaj N. et.al., Nature 501, 385390 (9), 2013 M. Faisal Shahid PCMD, ICCBS
- 2. 硫-barrel membrane proteins Essential for: Nutrient Transport Signaling Motility Survival
- 3. Gram Negative Bacteria BAM (硫-barrel assembly machinery) complex is responsible for biogenesis of 硫-barrel membrane proteins 4 components BamA BamB BamC BamD
- 4. Rationale for BamA structural study Mechanism for 留-helical membrane proteins is well established and acquainted but unknown for beta-barrel membrane protein(s)
- 5. What is known? In gram negative bacteria the Outer Membrane Proteins (OMPs) are synthesized in cytoplasm and transported across inner membrane into the periplasm by Sec translocon Further chaperones then escort them to inner surface of outer membrane Structures of BamB, BamB and BamC are available
- 7. What was done? Expression and purification of native BamA complex. X-Ray crystal structures of BamA from Neisseria gonorrhoeae (3.2 A属) and Haemophilus duceryi (2.91 A属) determined Both organisms are involved in sexually transmitted diseases (STDs), (N. gonorroheae in Gonorrhea and H. duceryi in Cancroid)
- 8. BamA structure at a glance 8-Outer surface loops 16 stranded 硫-barrel periplasmic domain Periplasmic domains termed POlypeptide TRanslocation Associated domains (POTRA domains)
- 9. Cloning/Expression PCR cloning in pET20b with PEL-B guide sequence For periplasmic proteins, soluble supernatant after cell pellet lysis, incubated with 2% Triton X-100 for 30 mins at room temp. Suspension then ultracentrifuged at 160,000g for 90 mins, and pellet re-suspended in Buffer-A of primary purification column. Insoluble suspensions were solubilized by addition of 5% Elugent, centrifuged at 265,000 x g for 60 mins. Supernatent filtered and loaded on Ni+2 affinity column, eluted with 250mM Imidazole, secondary purification performed on Sephacryl S300 columns.
- 10. Figure 1 | The structure of BamA from the BAM complex. a, The HdBamAD3 crystal structure in cartoon representation showing the b-barrel (green) and POTRA domains 4 and 5 (purple and blue, respectively). b, The NgBamA crystal structure showing the b-barrel (gold) and POTRA domains 15 (cyan, red, green, purple and blue, respectively). a b
- 11. c d C: A periplasmic (bottom) view of the NgBamA crystal structure. D: An alignment of the HdBamAD3 (green) and NgBamA (gold) crystal structures highlighting the structural conservation of the extracellular loops and secondary structural elements in loops (L) 4 and 6.
- 12. Structural features of BamA 硫-留-留-硫-硫 fold of POTRA domains is conserved POTRA domain of NgBamA located in close proximity of the periplasmic beta-barrel domain But tend to extend away in HdBamA3 structure
- 13. Barrel domain Each barrel domain contains 16 anti parallel 硫- strands First and last strands associate by hydrogen bonds Interior of barrel is almost empty Internal volume of ~13,000 A属
- 14. -------------------- -------------------- ------------------------------- ------------------------------ (a) and extracellular (b) view of an alignment of NgBamA and FhaC (grey, Protein Data Bank (PDB) code 2QDZ) illustrates conformational differences in the b-barrel and POTRA domains. In FhaC, the N-terminal a-helix (red) and loop 6 occlude the b-barrel preventing free diffusion across the outer membrane; however, in BamA this is accomplished by the extracellular loops that fold over the top of the barrel a b
- 15. Extracellular loops Extracellular loop eL4, eL6 and eL7 contribute substantially to the dome Minor contributions from 3L3 and eL8 eL4 has surface exposed 留-helix nearly parallel to membrane Strongly electropositive surface along eL3 and eL6
- 16. Alignment of the HdBamAD3 (green) and NgBamA (gold) crystal structures highlighting the structural conservation of the extracellular loops and secondary structural elements in loops eL6 eL3 eL4 eL5
- 17. Electrostatic surface representation of HdBamAD3 viewed from the extracellular face (a) and the periplasmic face (b)
- 18. POTRA domain conformations In NgBamA, POTRA5 sits proximally to barrel and interacts with periplasmic loops POTRA domains of HdBamA3 swings 70属 outward such that POTRA5 does not interact with periplasmic loops of the barrel loops in periplasm
- 20. Strand 16 of C-terminal Interface of strands 1 and 16 forms hydorgen bonding to close the barrel with 8 hydrogen bonds in HdBamA3 In NgBamA, structure of strand 16 interact using only 2 hydrogen bonds with strand 1 Allows BamA inter cavity access to lipid face of outer membrane at strand1:16 interface
- 21. Compared to HdBamAD3 (green), b-strand 16 is disordered and tucked inside the b-barrel of NgBamA (gold). Arrowheads indicate the location of the C-terminal strand in HdBamA (black) and NgBamA (red) FIRST OBSERVED EXAMPLE OF STRAND DESTABILIZATION OF CAVITY ACCESS THROUGH INERIOR OF BETA-BARREL Strand 16
- 22. BamA and FhaC homology model FhaC: Only source of structural information for membrane domain of Omp85 family Serves as dedicated toxin translocation pore in bacterial outer membrane Shares <13% sequence identity
- 23. Continued FhaC: Structure differs greatly with BamA RMSD for 硫-barrel domain is >10A属 Shear number for 硫-barrels is 20 (BamA =22) Extracellular Loops are in OPEN CONFORMATION Conformation of eL6 differs substantially with BamA eL6 contains VRGF/Y motif
- 24. Extracellular view of NgBamA (Gold) and FhaC alignment (Grey) N-termminal 留-helix (in FhaC) prevents free flow to solute (Extracellular loops show open conformation) In BamA, extracellular loops prevent free outward flow
- 25. NgBamA eL6 (Gold)contains a 硫-hairpin which is absent in FhaC (Grey) eL6 硫-hairpin is located 18 A属 above periplasmic surface of 硫-barrel in NgBamA (the loop bury inside periplasmic space in FhaC) -------------- ----------------------------------------- - ----------------------------------------- - VRGF/Y motif
- 26. eL6-VRGF/Y motif Distortion causes ablation of transport activity Interacts with beta strands 14-16A属 from periplasm R-658 (in HdBamA3) and R-660 (in NgBamA) interacts with E-696 & D713 in HdBamA and E692 & D713 in NgBamA Further stabized by F804,Q803,F802 FQF motif in strand 16 of beta-barrel
- 27. Homology modelling -barrel proteins have been most extensively studied in E. coli Homology model built for E. coli BamA Validation of model by mutagenesis
- 28. eL6-VRGF/Y motif V 660 R 661 F/Y 663 G 662 D 740 E 717 Homology model of EcBamA with conserved VRGF/Y motif F802 Q803 F804
- 29. Mutagenesis studies R661A mutant : Reduced colony growth VRGF>A : Leathal D740R: Leathal E717A/D740A double mutant: Minimal growth POTRA5 loops mutagenesis: No effect FQF mutations: No effect Potential disulphide bond in eL6: No effect Non-conserved loop (676-670) deletion: Reduced colony growth and slower doubling time
- 30. Phenotype growth effects Low expression levels and DegP up-regulation: R661A VRGF>A D740R E717A/D740A Interaction of R661 with barrel interior is important for proper function
- 31. Growth curve studies on mutants
- 32. Outer Membrane Distortion by Bam A HYPOTHESIS Compared to OMPs BamA 硫-barrel outer belt has greatly reduced hydrophobic C-termini This can destabilize local membrane environment
- 33. Proposed Mechanism of Protein Transport Molecular Dynamics stimulations used FhaC and Btu as control models for outer membrane
- 34. Continued Lipids close to C-termini of NgBamA has three fold decrease in order Membrane thickness near C-termini of NgBamA was 16A属 less than the opposite side of the barrel
- 35. Molecular dynamics analysis revealed that the b-barrel of NgBamA imparts a thinning of the membrane by 16A near strand b16 (centered at residue 788) when compared to the opposite side of the barrel (centered at residue 531), whereas no difference was observed for FhaC. Membrane disorder and increased distance suggest that: A major function of BamA in Bam comples is to prime membrane for OMP secretion
- 36. Gating Mechanism of BamA Stimulations demonstrated a LATERAL OPENING event in 硫-barrel of both structures via separation of first and last 硫-strands Separation between strand and POTRA5 oriented away from the barrel Distance ranged from 4A属 to 7.4A属 in HdBamA3 and 5A属-10A属 in NgBamA
- 37. Comparison between NgBamA (X-Ray crystal) 294K and MD-stimulated structure-310K Lateral Opening
- 38. Lateral Openings Only observed in three structures: FadL PagP OmpW All transport Hydrophobic molecules A closing event was also observed in MD- stimulations with interval of 1袖 second
- 39. Conclusion BamA can perturb outer membrane by: Reduced hydrophobic surface near 硫-strand 16 resulting in decreased lipid order and membrane thickness Transient separation of 硫-strands 1 and 16 With PORTA domains, highly dynamic membrane environment is created by BamA in immediate vicinity of Bam Complex Some 硫-barrels can be folded in periplasm before insertion into outer membrane (insertion mechanism unclear)
- 40. Possible Mechanism of BamA mediated protein entry Use of hypothetical conformation switch of eL6, POTRA5 and lateral opening event OR OMPs may be trafficked into close proximity of outer membrane via interactions with POTRA5 domain to transiently destabilizing outer membrane patch to make room for protein insertion
- 41. Thank you
- 42. Questions?