Lecture 1 OMP biogenesis

Question 1

Name 4 characteristics of the inner membrane of Gram-negative bacteria

Answer 1

• Phospholipid bilayer
• Symmetric
• Integral membrane proteins (interactions on either side of the membrane)
• Peripheral lipoproteins (attached to the membrane, but only on one side)

Question 2

Name 5 characteristics of the OM of Gram-negative bacteria

Answer 2

- Outer membrane (not a phospholipid bilayer)
• Phospholipid inner leaflet
• LPS in outer leaflet
• Asymmetric (bc different leaflets)
• Integral membrane proteins  OMPs
• Peripheral lipoproteins

Question 3

Characteristics of IMP?

Answer 3

• are alfa-helical
• Transmembrane helices (TMs) act as membrane anchors
• (1 - 10 TMs)
  • AAs (amino acids) in TMs are hydrophobic and contact acyl chains of phospholipids
  • (~20AA/TM)

Question 4

Characteristics of OMP?

Answer 4

• folded into a barrel
• even number of beta-strands (8-24)
• strands 10 to >40 amino acid residues
− difficult to predict (from genes)

Question 5

What is the build of OMP and how are the side chains positioned?

Answer 5

• Built op of anti-parallel beta strands (are amphipathic, both hydrophobic and -philic)
• amino acid side chains stick out from surface of beta sheet. If side chains are hydrophobic they are at the outside, hydrophilic inside.
• Extra domains (periplasmic) are always found at the N-terminus

Question 6

What do the beta-barrels of OMPs look like?

Answer 6

• Mono- or multimeric
− often trimers
• small loops at periplasmic side
• Long loops extending from the cell surface
− often functional ( e.g. as binding site, or used in gating )

Question 7

Why are OMPs able to pass the cytoplasmic membrane to the periplasm?

Answer 7

Sec-machinery considers OMPs as soluble proteins -> OMPs have amphipathic beta-strands, which are not that hydrophobic, and can pass the cytoplasmic membrane to the periplasm.

Question 8

What proteins do not pass the membrane and are placed in the cytoplasmic membrane?

Answer 8

Proteins containing alpha helices that are hydrophobic are placed laterally in the cytoplasmic membrane. Would not pass the membrane.

Question 9

Name examples of OMP’s

Answer 9

• Trimeric Porins
Channels for uptake small molecules/nutrients
• Specific transporters
E.g. FhuA (iron ion receptor), recognize specific molecules
• Enzyme for modification of substrate (LPS). No pore. Modification of substrate
Often shorter beta-sheets. Such close contact of inward side chains that water cannot go through anymore, no hydrophobic channel inside
• Enzyme that anchors OM to peptidoglycan. No pore.
E.g. OmpA

Question 10

Via what secretion system are OMPs transported through the inner membrane to the OM?

Answer 10

Sec

Question 11

How do OMPs cross the inner membrane via Sec?

Answer 11

• N-terminal signal peptide (hydrophobic alpha helical stretch for targeting). Remaining part is not that hydrophobic.  OMPs enter periplasm unfolded. Signal peptide is cut off. Chaperones keep them in a transport competent state and prevent aggregation in periplasmic space.

Question 12

What are the two functions of chaperones in the periplasm?

Answer 12

• Prevent aggregation
− Shield hydrophobic parts when protein starts to fold. Hydrophobic site is not compatible with the hydrophilic periplasm.
• Assist in folding
− Barrel is formed in periplasm prior to insertion in OM

Question 13

What chaperones help with OMP insertion? What is their function?

Answer 13

Skp, SurA
• protect against preliminary degradation
> Encage the OMP
DegP
• Has protease activity, 
degrades unfolded OMPs
> part of rpoE stress response

Question 14

Skp: characteristics?

Answer 14

• Homotrimer
• Binds unfolded OMPs
• Does not bind folded OMPs
• Forceps-like arms
• Prevents aggregation of OMPs
• No role in folding

Question 15

SurA characteristics?

Answer 15

Survival protein A (SurA)
• When mutated (knock-out): less OMPs produced, less OMPs assembled into OM
• Binds peptides rich in aromatic residues (which you find at rings of beta-barrel structures)
• Binds preferentially unfolded OMPs (better recognition of aromatic peptides)
• Helps formation of beta-barrel structure
- Folding chaperone
- Contains “PPI” activity

Question 16

What is PPI activity?

Answer 16

“Peptidyl-prolyl cis/trans isomerase”

Changes conformation of prolines, which have a cis or trans confirmation and can be found in protein backbone. Prolines often positioned in turns and kinks. Changes protein conformation.

You find prolines mostly in the short turns in the periplasmic loops.

Question 17

When are chaperones upregulated when the cell encounters stress?

Answer 17

With stress that interferes with the integration of OMPs in the OM and stress that is related to the intergrity of the OM

Question 18

What happens when surA and Skp are knocked out together?

Answer 18

• SurA and Skp can be knocked out, but NOT together.
  = Synthetic lethality ( type of genetic interaction where the combination of two genetic events results in cell death or death of an organism.)
  Needed for efficiency and protection.

Question 19

What happens when there is stress in the cell regarding rpoE plasmid?

Answer 19

RPoE = Sigma e stress = whether proteins accumulate in the periplasm/not fold correctly. Also induced when soluble periplasmic proteins are accumulating

Question 20

What is the response of RpoE?

Answer 20

Overproduction of DegP protease.

Question 21

What does DegP do and how?

Answer 21

DegP: periplasmic protease

Forms a ring-like structure of 4 monomers -> stack of rings (12-24) on top of each other.
Cavity: degradation of not properly folded OMPs
Also chaperone -> can assist folding of beta-barrels
[not present in all bacteria]

Question 22

What does the Bam-complex do?

Answer 22

Assists in the final folding of the beta-sheet into a beta-barrel. Inserts barrel into the OM.

Question 23

What kind of gene did they look for when searching the candidates for OMP insertase?

Answer 23

• Conserved among Gram-negatives, mitochondria and chloroplasts
• Must be an essential gene

Question 24

They found OMP85. Why was this a good candidate?

Answer 24

Essential
Located in OM
In operon with skp (OMP chaperone) and lpxA (LPS biosynthesis). Both linked to OM biogenesis

Question 25

OMP85: essential gene | > Could not be knocked out, how to study?

Answer 25

Construct a conditional mutant. 1. Introduce inducible copy of omp85 gene on a plasmid. 2. Knock-out chromosomal copy. Mutant carrying this knock-out is still viable because it has the plasmid that contains the WT gene 3. Cells get depleted of Omp85 protein in absence of the inducer. When introducing cells to inducer-less medium, there is a window in which you can study them.

Question 26

How to check for correct OMP-assembly?

Answer 26

Folding of OMPs can be monitored by SDS-PAGE • Heat-modifiability: Take two samples of a cell envelope, one which is denatured by heating, one at room temperature. Monitor whether presence of Omp85 led to folded or unfolded OMP at room temperature and denatured cell envelope.

Question 27

Characteristics of OMP regarding denaturation & stability?

Answer 27

 beta-barrel very stable  resists denaturation by chemical SDS at room temp.  folded form runs faster on SDS-PAGE

Question 28

Depleted Omp85 protein lead to unfolded OMPs. Conclusion?

Answer 28

Assembly OMPs into the outer membrane requires Omp85, renamed: BamA (Bam: Beta-barrel Assembly Machine)

Question 29

BamA was found in complex with..?

Answer 29

With 4 peripheral lipoproteins. BamB-E. BamD also essential, other components not.

Question 30

What was an indication that Bam complex could push OM proteins towards the beta barrel?

Answer 30

Crystal structure; 2 structures. They had differently distributed POTRA domains, as if there was some kind of movement. (POTRA domains are beneath the beta barrel)

Question 31

What happens when deleting POTRA domains?

Answer 31

POTRA domains deleted from BamA • deletion of all is lethal • Decreasing number decreased efficiency of insertion • Effect more prominent on larger OMPs (22-stranded barrels)

Question 32

Function of POTRA domains?

Answer 32

The lipoproteins include lots of beta-strands. Beta-strands align and interact with the pre-mature beta-barrel proteins: a process that is called beta-augmentation. Helps align beta strands next to each other so it helps to form the sheet.

Question 33

What does BamA do with the membrane to make the uptake of barrels easier?

Answer 33

When looking at the aromatic rings, the membrane looks as is it is ‘squeezed’ at the backside. In vitro folding of OMPs goes better when membrane is thin. Thinner membrane less stable, more movement lipids  easier for uptake barrels

Question 34

What else indicates something is in the lumen of BamA and then pushed into the membrane?

Answer 34

BamA has a lateral gate. First and last strand of beta strand positioned differently than in normal beta sheet proteins and they are shorter. Open vs closed state.

Question 35

How did they find out the lateral gate is important for the process?

Answer 35

Cross-linking the gate by two cysteines • Form disulfide bond. Could this disulfide bond protein replace the normal protein? Effect: • BamA inserted in OM • BamA not able to complement wt-BamA (wt-BamA gene controlled by Arabinose-inducibe promoter)

Question 36

What is the role of POTRA domains regarding the lateral gate?

Answer 36

 POTRA domains hinge the lateral gate to close and open.

Question 37

What are the current models for OMP insertion?

Answer 37

1. BamA assisted model - provides a dynamic lipid bilayer - OMP folds spontaneously POTRA domains - bc of lipid movement: insertion takes place 2. BamA-budding Lateral gate is the place for beta-barrel folding and sideways translocating into the lipid bilayer 3. Barrel-elongation POTRA domains receive OMP, bring beta-strands into contact with the lateral gate, which helps to fold the sheet into the final barrel confirmation, then it is pushed into the OM.

Question 38

How is the process of OMP insertion energized?

Answer 38

interaction of the OMP + BamA = confirmational change. Lower folding energy -> difference in energy = used to help bamA change confirmation and push protein in lipid bilayer

Question 39

How does BamA recognize an OMP?

Answer 39

C-terminal targeting motif is important (deletion = aggregation in periplasm) in OMP (aromatic residues at the end, sort of conserved sequence)

Question 40

You can show pore activity with BamA conductivity assay. How does this work?

Answer 40

Two compartments: small hole in between. Lipid bilayer shutting off hole. Two electrodes: try to run a current, hydrophobic barrier prevents formation of a current. Then, to one compartment you can add the BamA protein, sometimes it will spontaneously insert itself into the lipid bilayer. Then you get a pore when unfoled OMP with signal sequence is added: current will run. Sometimes channels open and close: fluctuation in current. Pore does not open when adding OMP without signal sequence.

Question 41

Overview

Answer 41

1. Integral OMPs are mostly beta-barrels 2. Folding/assembly starts in periplasm 3. Correct folding helped by chaperones 4. BamA has role in membrane insertion and targeting • lateral gate • POTRAs • Lipid deformation 5. C-terminus OMP contains Bam-targeting motif