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 ShahidPCMD, ICCBS

β-barrel membrane proteins

• Essential for:– Nutrient Transport– Signaling – Motility– Survival

Gram Negative Bacteria

• BAM (β-barrel assembly machinery) complex is responsible for biogenesis of β-barrel membrane proteins

• 4 components– BamA– BamB– BamC– BamD

Rationale for BamA structural study

• Mechanism for α-helical membrane proteins is well established and acquainted but unknown for beta-barrel membrane protein(s)

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

The Periplasmic Space

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)

BamA structure at a glance

• 8-Outer surface loops

• 16 stranded β-barrel periplasmic domain

• Periplasmic domains termed POlypeptide TRanslocation Associated domains (POTRA domains)

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.

Figure 1 | The structure of BamA from the BAM complex. a, TheHdBamAD3 crystal structure in cartoon representation showing the b-barrel(green) and POTRA domains 4 and 5 (purple and blue, respectively). b, TheNgBamA crystal structure showing the b-barrel (gold) and POTRA domains

1–5 (cyan, red, green, purple and blue, respectively).

a b

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.

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

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°

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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 inthe b-barrel and POTRA domains. In FhaC, the N-terminal a-helix (red) andloop 6 occlude the b-barrel preventing free diffusion across the outermembrane; however, in BamA this is accomplished by the extracellular loopsthat fold over the top of the barrel

a b

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

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

Electrostatic surface representation of HdBamAD3viewed from the extracellular face (a) and the

periplasmic face (b)

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

HdBamAΔ3 NgBamA

POTRA 5

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

Compared to HdBamAD3 (green), b-strand 16 is disordered and tuckedinside 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

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

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

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

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)

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VRGF/Y motif

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

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

eL6-VRGF/Y motif

V 660R 661

F/Y 663

G 662

D 740

E 717

Homology model of EcBamA with conserved VRGF/Y motif

F802Q803F804

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

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

Growth curve studies on mutants

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

Proposed Mechanism of Protein Transport

• Molecular Dynamics stimulations used

• FhaC and Btu as control models for outer membrane

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

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”

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

Comparison between NgBamA (X-Ray crystal) 294K and MD-stimulated structure-310K

Lateral Opening

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

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)

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

Thank you

Questions?