Lecture 3: Structural biology of bacterial secretion systems

Question 1

What did protein machines in the gram-negative cell envelope evolve from?

Answer 1

Adapted/evolved from macromolecular structures present on the bacterial surface
− Pili (II) / Flagella (III) / Conjugation systems (IV)

Question 2

What type of secretion systems use a two-step secretion?

Answer 2

T2SS and autotransporters (T5SS)

Question 3

How is two-step secretion performed?

Answer 3

• cytoplasmic membrane: enter via Sec/Tat using a signal peptide, to the periplasm
• periplasmic chaperones guide the proteins toward the secretion system in an unfolded, inactive state so that they only fold when being released into the extrac. milieu

(two-step because substrates are first translocated across the bacterial inner membrane; once in the periplasm, substrates are targeted to one of the secretion systems that mediate transport across the outer membrane and released outside the bacterial cell)

Question 4

How is energy for this transport generated as there is no energy at OM/periplasmic space?

Answer 4

Energy is generated by the IM part of the complex, ATP is hydrolyzed, energy for building up machinery and confirmational changes that help transport

Question 5

What does one-step secretion mean? What type of secretion systems use this?

Answer 5

One-step secretion: transported directly to the outside and sometimes into the host environment (T3SS, T1SS, T4SS)

Question 6

How is the substance transported and targeted to the right machinery?

Answer 6

Motifs that recognize machinery/chaperones in cytosol that guide substrate or combination of both

Question 7

Crystallography: how is it done?

Answer 7

Crystallography classical route to determine protein structure
• Purify protein (folded) in large amounts
• Prepare crystals using precipitation (trial and error)
• Obtain diffraction pattern using X-ray
• Determine structure from pattern (density map) -> turn into 3D cartoon-like representation

Question 8

Multiple crystals of same proteins may reveal different possible
confirmations. Why?

Answer 8

• presence of substrates

* presence of ligands, cofactors, inhibitors

Question 9

Why is crystallography not very suitable for membrane proteins?

Answer 9

However, crystallography is not very suitable for membrane proteins:
• Hydrophobic surface
• insoluble in buffers / crystallization solutions (rely on salts)
• Difficult to purify from membranes
‒ Solubilize membranes using detergents
‒ Production low
‒ Proteins tend to aggregate
> Difficult to obtain high concentrations

Question 10

What is an alternative to determine the structure of MP?

Answer 10

Electron Microscopy can be used

Question 11

Electron microscopy: electron beams detect electron-dense material. Biological samples often need staining with heavy metals. What is a way to prevent doing this?

Answer 11

• Cryo-EM; freezing at ultra-low temperatures to 
- prevents radiation damage to samples, 
- no staining needed
- improves density
Suitable for bacterial cells, sectioned/sliced samples and purified complexes
• Lower concentrations required
• Image averaging improves resolution
• Samples can be tilted  3D tomography

Question 12

How does cryo-EM go?

Answer 12

1. Purified proteins/complexes are loaded on to a EM grid
   • membrane proteins solubilised in detergents
2. similar images are grouped -> different orientations
3. when similar images are averaged: better resolution

Question 13

• cells can be frozen quickly: Plunging in liquid ethane
> Under extreme cold and high pressure
• followed by sectioning
(what is that called?)

Answer 13

Tomography

Question 14

cryo-EM tomography of Type III complex: how was it done and what are advantages of this technique?

Answer 14

• prepare vitrified cells
• perform tomography (100nm slices)
• image with EM
• do averaging of identified complexes

Advantage:
• no purification
• less loss of complex
components

Question 15

How is the T1SS built up?

Answer 15

IM: ABC transporter (atp hydrolysis part: contains motifs that are called ABC: ATP binding cassettes. Sequencing: characteristic for T1SS)
− TM-helices
− ATP-binding & hydrolysis
− IM transport channel

Periplasmic adaptor protein
− TM-helix as anchor in IM
− Fuses IM-compex to OMP

OM: b-barrel channel
− ~30Å channel (quite large)

Question 16

What does the T1SS secrete?

Answer 16

Proteins; toxins & enzymes
• Polysaccharides; Capsule
Components

Question 17

T1SS: trimeric beta-barrel in the OM. Funnel of alfa helices in periplasm. Strands donated by 3 subunits. How does the open/closing of the funnel take place?

Answer 17

By twisting of the alfa helices.

Question 18

What is the opened/closed state dependent of?

Answer 18

Open and closed state
– Binding of substrate to ABC transporter
– ATP-dependent translocation
– Substrate unfolded during transit

Question 19

T1SS = multidrug efflux pump. Structure solved by cryo-Em. Which components are system specific and which are not (in E.coli)?

Answer 19

In E. coli: TolC used for both systems as OMP channel. —>
System-specific: periplasmic adaptor protein and ABC transporter (specific IMP for transport: distinct in multidrug efflux pumps). Pumped-out by Efflux pump: Antibiotics, Harmfull chemicals (SDS)

Question 20

What is the T3SS involved with? What does it do?

Answer 20

Type III secretion system involved in interaction with host cells.
They bind to a host cell but don’t go inside. Yersiniae replicate outside host cells, but escape phagocytosis.

Question 21

T3SS is used for virulence in Yersinia species. How is this performed?

Answer 21

Yersinia outer proteins (Yops) modulate host cell processes (interfere with signal transduction that regulates host skeleton): actin shields yersinia. Membrane structures formed around it = intimate attachment.

• Secreted via Type III (looks like a needle)
Then, different enzymes are inserted: induction apoptosis (for nutrients) and anti-inflammatory responses in macrophages

Question 22

How is the needle length controlled? By what molecule? What else does this molecule do?

Answer 22

YscP controls needle length

When YscP was deleted the length of the needle became different and undefined

YscP is a molecular ruler: ruler length decided by repeats in protein. Change number -> length.

Serves as a plug to prevent premature transport & a ruler to determine the length.

Question 23

What length is required for the needle in order to transport?

Answer 23

Length of the needle coincides with the length of the complex of the adhesin (YadA adhesin of Yersinia in this case) which is binding to a receptor. (=length bacterium-host cell) If its bound, certain length, needle matches. Optimal distance between bacterium and cell required. No match, no contact, no transport.

Question 24

T6SS: First identified in V. cholerae. one or two step?

Answer 24

One-step.

Question 25

How was T6SS identified?

Answer 25

1. V cholera: gene clusters with two homologues of a T4SS.
2. Multiple bacterial species carried a similar genomic island
3. Shown to be a seperate secretion system

Question 26

What is T6SS used for? How did they find out?

Answer 26

• Transposon insertions in the genomic islands encoding for T6SS attenuate (inhibit) virulence
• Hcp/VrgG proteins in vibrio supernatant. These proteins are used for virulence.

Type VI: kill other bacteria/organisms competing for niche

Question 27

Cryo EM and homologous searches were done for secreted components. What was found out?

Answer 27

Structural similarities between gpVn and Hcp1 (secreted by V cholera). gpVn is a protein encoded by bacteriophages -> in tail structures. Part that connects to the host cell.
Type 6 an inverted bacteriophage injection machine.

Question 28

How does T6SS inject into a neighboring cell?

Answer 28

1. Build up of tube: Inner tube + outer tube
2. Signalling
3. Outer tube = contracting and ejects the inner tube, brings tip complex in neighboring cell
4. Degradation

Question 29

How does the bacteria compete for niches: how does it kill opponents?

Answer 29

Proteins are inserted into the periplasm that attack peptidoglycan, make cell lose its structure.
Also, membrane inserting peptides that create pores and RNases and DNases. Cell is pierced, lysed, genetic content is destroyed.

Bacteria that use the model are pathogenic and usually colonize niches where there is already a resident microflora (intestine, skin, lungs..) Need space by attacking bacteria already there.

Question 30

What was solved by crystollagraphy and what by cryo-EM of T1 system?

Answer 30

Type I trimeric OMP structure solved by crystallography, complex by cryo-EM