L17 : Secretion and Polymerising Machinery Flashcards
What is the role of pilus in gram neg bacteria?
Involved in recognition and attachment of bacteria to host tissues
Type P - targets kidneys (pyelonephritis)
Type 1 - targets bladder (cystisis)
Main determinants of UTIs
What is the role of the Pap operon?
Pap gene cluster operon encodes subunits of the P pilli
What is the role of the chaperone and path of subunits?
Subunits are produced on cytosolic side of inner membrane within bacteria
Unfolded polypeptides pass through SecYEG transporter
Taken up by chaperone papD, which assists in folding subunits
Subunits and chaperone remain in a stable binary complex within periplasm
What is the role of the usher?
PapC is outer membrane assembly platform and orchestrates pilus biogenesis
Each chaperone-subunit complex goes to papC to engage in cycle of subunit incorporation
What is order of assembly of subunits?
Assembly of pilus subunits is a highly ordered process mediated by usher
PapG-papD has highest affinity for usher to able to bind and activates usher
Cycle continues with incorporation of papF and then ~10-20 papE
Binding of junction subunit papK completes tip fibrilium and triggers polymerisation o papA to form pilus rod
papH is the termination subunit, ending pilus biogenesis
Describe the structure of the subunits
All subunits have similar structure
Pilus subunits lack beta strand G (sits antiparallel to F) so possess less stability
Missing seventh strand leaves large groove at surface of protein
What is the mechanism of donor strand completion by chaperone?
Truncated structure very unstable on its own and prone to aggregation
N-terminal domain of chaperone has strand that can insert into groove of subunit, stabilising and completing the subunit fold
What is the structural basis of donor strand exchange mechanism?
N-terminal extension from next pap subunit fills gap of previous
Proceeds one subunit at a time, with chaperone dissociating each time to allow the next subunit to complete DSE
Following DSE, fibre is formed by string of typical Ig folded subunits
What are the molecular details of DSE reaction?
Mass spec revealed a zip-in-zip-out mechanism
N-terminal extension introduces P5 residue at C-terminus into the P5 pocket of the chaperone subunit
Nt extension ‘zips in’ and forms ternary complex
Chaperone strand ‘zips out’ and dissociates
Why is papH the termination subunit?
Studies involving deletion and overexpression are consistent with papH being involved in termination of pilus biogenesis
papH does not have P5 pocket so is unable to insert an N-terminal extension, terminating biogenesis
What is the molecular basis for subunit ordering?
PapD-papG has highest affinity for usher so begins pilus biogensis
In vitro DSE assay set up and reaction monitored using mass spec
Results show high degree of specificity of Nt extensions for respective groove so only correct Nt extension able to react
What is known about the usher?
Outer membrane assembly platform and site of subunit polymerisation
Outer membrane secretion pore, transporting subunits to surface of bacterium
Large protein - 24 stranded beta barrel (809 AAs)
What is the domain structure of usher?
N-terminal domain essential in subunit recruitment and responsible for binding chaperone-subunit complexes
Both translocation and C-terminal domain structures are unknown
How was crystal structure of FimD:FimC:FimH complex created?
Coexpressed all protein (FimD usher + FimC chaperone + FimH adhesin)
Complex purified from outer membrane using detergent
Molecule then crystallised to determine structure
What did the crystal structure show about mechanism of usher?
FimC:FimH bound to CTDs of usher
Shows 2 binding sites for chaperone:subunit complexes
First chaperone transferred to second binding site after DSE
NTD in FimD:FimC:FimH complex ideally positioned to bind next incoming chaperone-subunit complex FimC:FimG
Binding of FimC:FimG to NTD positioned Nte of FimG for DSE with FimH
Process repeats to carry out pilus biogenesis
