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.

1. Biogenesis of β-barrel
Membrane Proteins
Simplified elaboration of beta barrel
protein incorporation mechanisms in
cells as cited under:
Noinaj N. et.al.,
Nature 501, 385–390 (9), 2013
M. Faisal Shahid
PCMD, ICCBS

2. β-barrel membrane proteins
• Essential for:
– Nutrient Transport
– Signaling
– Motility
– Survival

3. Gram Negative Bacteria
• BAM (β-barrel assembly machinery) complex
is responsible for biogenesis of β-barrel
membrane proteins
• 4 components
– BamA
– BamB
– BamC
– BamD

4. Rationale for BamA structural study
• Mechanism for α-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

6. The Periplasmic Space

7. What was done?
• Expression and purification of native BamA
complex.
• X-Ray crystal structures of BamA from Neisseria
gonorrhoeae (3.2 A°) and Haemophilus duceryi
(2.91 A°) 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 β-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
1–5 (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
• β-α-α-β-β 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 HdBamAΔ3 structure

13. Barrel domain
• Each barrel domain contains 16 anti parallel β-
strands
• First and last strands associate by hydrogen
bonds
• Interior of barrel is almost empty
• Internal volume of ~13,000 A°

14. --------------------
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-------------------------------
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(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 α-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 HdBamAΔ3 swings 70°
outward such that POTRA5 does not interact
with periplasmic loops of the barrel loops in
periplasm

19. HdBamAΔ3NgBamA
POTRA 5

20. Strand 16 of C-terminal
• Interface of strands 1 and 16 forms hydorgen
bonding to close the barrel with 8 hydrogen
bonds in HdBamAΔ3
• 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 β-barrel domain is >10A°
– Shear number for β-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 α-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 β-hairpin
which is absent in FhaC
(Grey)
eL6 β-hairpin is located 18 A°
above periplasmic surface of
β-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° from periplasm
• R-658 (in HdBamAΔ3) 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
• Β-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 β-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° 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 β-barrel of both structures
via separation of first and last β-strands
• Separation between strand and POTRA5
oriented away from the barrel
• Distance ranged from 4A° to 7.4A° in
HdBamAΔ3 and 5A°-10A° 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 1µ second

39. Conclusion
• BamA can perturb outer membrane by:
– Reduced hydrophobic surface near β-strand 16 resulting in
decreased lipid order and membrane thickness
– Transient separation of β-strands 1 and 16
• With PORTA domains, highly dynamic membrane
environment is created by BamA in immediate vicinity
of Bam Complex
• Some β-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?