Lecture 18 - Pilus Biogenesis Flashcards
What is the role of Type 1 and Type P Pilli?
Type P pil target the kidneys
Type 1 Pili target the bladder
What is the role of the Pap operon?
The Pap gene cluster is a cluster of genes that encode the machinery of the P pili.
What proteins are involved in the assembly of the pilus?
PapD and the outer membrane usher. and the subunit proteins.
Explain the journey of the usher and various subunits.
the usher and subunits are produced on the ribosome.
Then they are transported through the inner membrane into the periplasmic space via the transporter YEG.
The proteins cannot fold on their own, so they associate with the chaperone papD.
The subunits are then left in a binary complex with the chaperone.
What is the role of the usher?
The usher is transported to the outer membrane, and its role is to orchestrate the assembly of the pilus.
What is the order of assembly of the subunits?
The red protein needs to be assembled first, so the papG-papD complex is transported to the usher first.
Then the papF-papD (orange protein) complex is transported to the usher, where papF reacts with papG, and the chaperone on papG is released, to allow assembly.
The same cycle continues with multiple units of PapE (yellow protein) and 1 unit of PapK (green), and 1000s of units of PapA (blue) respectively.
The PapA forms a pilus rod
Then PapH reacts with PapA, triggering termination of the assembly
Explain the structure of the chaperone PapD and subunits
The chaperone is a two-domain structure.
All the subunits have a very similar structure.
The subunit has a groove that goes through, caused by the lack of a beta strand.
What issues does the groove on the subunits cause, and how is this fixed?
The groove causes the hydrophobic core of the subunit to be exposed which can lead to protein aggregation.
The chaperone donates a ‘G-strand’ to the subunit to ‘rescue’ the subunit.
This is called donor strand complementation.
What replaces the Gap in the groove on the subunit once the chaperone departs?
All subunits have an N-terminus extension.
When the chaperone dissociates, and another subunit is bought for assembly, the N-terminus extension provides the ‘G-strand’.
This is how subunits polymerise with each other.
The change of donor is called donor strand exchange.
What are the molecular details of the donor strand exchange reaction?
Donor strand exchange proceeds through a zip in-zip out mechanism.
A P5 residue on the C-terminus fits into a P5 pocket on the subunit.
A ternary complex of the chaperone, subunit, and peptide is formed.
This is backed up by Mass Spec
What are the molecular details of the pilus biogenesis termination reaction?
PapH is the only subunit that does not have a P5 pocket.
Therefore no subunits can react with PapH, and donor strand exchange cannot occur.
What are the molecular details of subunit ordering?
The affinity for the usher is highest for PapD-PapG, so it is transported first.
An in vitro donor strand exchange assay was set up, and the reaction was monitored via mass spec.
PapD-PapG has highest affinity for PapD-PapF
PapD-PapA has highest affinity for PapD-PapA.
Explain the domain structure of ushers.
N-terminal domain is responsible for binding chaperone-subunit complexes, and plays an essential role in subunit recruitment.
Structure of translocation domain unknown.
Function of C-terminal domain unknown.
