Lecture 3: Structural biology of bacterial secretion systems Flashcards
What did protein machines in the gram-negative cell envelope evolve from?
Adapted/evolved from macromolecular structures present on the bacterial surface
− Pili (II) / Flagella (III) / Conjugation systems (IV)
What type of secretion systems use a two-step secretion?
T2SS and autotransporters (T5SS)
How is two-step secretion performed?
- 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)
How is energy for this transport generated as there is no energy at OM/periplasmic space?
Energy is generated by the IM part of the complex, ATP is hydrolyzed, energy for building up machinery and confirmational changes that help transport
What does one-step secretion mean? What type of secretion systems use this?
One-step secretion: transported directly to the outside and sometimes into the host environment (T3SS, T1SS, T4SS)
How is the substance transported and targeted to the right machinery?
Motifs that recognize machinery/chaperones in cytosol that guide substrate or combination of both
Crystallography: how is it done?
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
Multiple crystals of same proteins may reveal different possible
confirmations. Why?
- presence of substrates
* presence of ligands, cofactors, inhibitors
Why is crystallography not very suitable for membrane proteins?
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
What is an alternative to determine the structure of MP?
Electron Microscopy can be used
Electron microscopy: electron beams detect electron-dense material. Biological samples often need staining with heavy metals. What is a way to prevent doing this?
• 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
How does cryo-EM go?
- Purified proteins/complexes are loaded on to a EM grid
• membrane proteins solubilised in detergents - similar images are grouped -> different orientations
- when similar images are averaged: better resolution
• cells can be frozen quickly: Plunging in liquid ethane
> Under extreme cold and high pressure
• followed by sectioning
(what is that called?)
Tomography
cryo-EM tomography of Type III complex: how was it done and what are advantages of this technique?
- prepare vitrified cells
- perform tomography (100nm slices)
- image with EM
- do averaging of identified complexes
Advantage:
• no purification
• less loss of complex
components
How is the T1SS built up?
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)