1
L23 Protein Functions as a Chaperone Docking
Site on the Ribosome

• Kramer, G., et. al. (2002) Nature 419 171-174

Presented by Michael Evans Department of
Chemistry and Biochemistry University of Notre
Dame Notre Dame, IN 46616
2
Overview

• Introduction to chaperones
• Experiments and Results
• Conclusions
• Future Work

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Chaperones and Folding

• Newly synthesized polypeptides must fold to
  native conformation in crowded environment of the
  cell
• Chaperones help many to avoid aggregation
• Bind to exposed hydrophobic regions
• PPIase activity
• ATP dependent binding
• Maintain conformational flexibility

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Chaperone Pathway in Bacteria
Hartl, F.U. and Hayer-Hartl, M. (2002) Science
295 1852-1858
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Trigger Factor (TF)

• First bacterial chaperone to see nascent
  polypeptide
• Has PPIase activity, but recognizes hydrophobic
  residues
• Function overlaps with DnaJ/DnaK chaperones
• N-terminal domain mediates binding to 50S subunit
  of ribosome

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Significance

• Explain coupling of synthesis to folding
• Eukaryotic parallels
• No TF
• Other chaperones interact with ribosome
• SRP study

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A Few Questions

• What part of TF is important for interaction with
  the ribosome?
• Which ribosomal protein(s) and/or RNA does TF
  interact with?
• Must TF bind ribosomes to interact with nascent
  chains?
• Is ribosomal association required for TFs
  participation in protein folding?

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TF Signature

• Alignment of TF homologues revealed 17 conserved
  residues
• Completely conserved G-F-R-X-G-X-X-P motif--the
  TF signature
• TF signature located in unstructured region
• Could be surface-exposed and contribute to
  ribosome interaction

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TF Signature and Mutants
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TF Signature Mutants

• FRK/AAA should show reduced association with
  ribosomes

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FRK/AAA Mutant Association with Ribosomes

• Incubated FRK/AAA with ribosomes from ?tig E.
  coli
• Ribosomes separated from unbound protein by
  centrifugation
• SDS-PAGE of pellet (ribosome) and supernatant
  (unbound protein)

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FRK/AAA Mutant Association with Ribosomes

• Increased amount of FRK/AAA in supernatant
  relative to wt TF incubated with ribosomes

S Supernatant P Ribosome Pellet
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TF Signature Mutants

• D42C replace Asp with Cys to allow attachment of
  crosslinking reagent
• BPIA is UV activatable
• Attacks C-H bonds, so will react with ribosomal
  proteins and RNA

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D42C Mutant Association and Crosslinking with
Ribosomes

• Couple TF D42C to BPIA
• Incubate with ?tig ribosomes
• Activate BPIA by UV irradiation
• Separate ribosome-protein complexes as before by
  centrifugation
• SDS-PAGE to resolve crosslinking products

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D42C Mutant Association and Crosslinking with
Ribosomes

• Two products, 68 kDa and 75 kDa
• RNase A treatment does not affect mobility of
  products
• Trypsin digestion followed by ESI-MS to identify
  cross-linked proteins
• 68 kDa TF L29
• 75kDa TF L23

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Interaction is Specific

• Add 2.5 M excess of either wt TF or FRK/AAA to
  compete with D42C-BPIA during crosslinking
• wt TF results in decrease of both crosslinking
  products
• FRK/AAA does not decrease yield of crosslinking
  products
• Crosslinking products are a result of a specific
  TF-ribosome interaction

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L23 and L29

• Both proteins of the large subunit
• In direct contact with each other
• Located next to the exit tunnel
• Does TF associate directly with one or both?

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L23 and L29 Deletion Mutants

• Strategy replace ORF with kanamycin resistance
  cassette

Adapted from Datsenko, K.A., and Wanner, B.L.
(2000) Proc. Nat. Acad. Sci. 97 6640-6645
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L23 and L29 Deletion Mutants

• Two mutants produced
• ?rpmCkan, deletion of L29 gene
• ?rplWkan, deletion of L23 gene
• ?rpmCkan grows, but slightly slower than wt
• ?rplWkan requires presence of pL23 for growth

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L23 and L29 Deletion Mutants

• ?rplWkan growth dependent on IPTG induction of
  pL23
• L23 mutant is also viable

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L29 and TF Binding

• Purify ribosomes from ?rpmCkan under high salt
  conditions
• Does TF remain bound to ribosomes without L29?
• Can TF rebind ribosomes without L29?

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TF Remains Associated to L29-Deficient Ribosomes

• SDS-PAGE of isolated ribosomes
• Control is from ?rplW cells with wt L23 from
  plasmid
• TF remains associated with L29-deficient ribosomes

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TF Can Rebind to L29-Deficient Ribosomes

• SDS-PAGE of ribosome-TF pellet and supernatant
• Control is from ?rplW cells with wt L23 from
  plasmid
• TF associates with L29-deficient ribosomes

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L23 Deletion and Mutants

• L29 is not required for TF binding, but what
  about L23?
• ?rplW mutants are nonviable, but pL23 rescues
• What part of L23 is important for binding?

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L23 Region 1 and 2 Mutants

• Criteria for interaction
• residue is surface-exposed
• Conserved among bacterial L23s
• Two regions identified

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L23 Region 1 and 2 Mutants

• Region 1 E18A, E18Q, VSE/AAA
• Region 2 E52K, FEV/AAA
• All mutant L23s complement ?rplW

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L23 Mutants and TF Binding

• Only region 1 mutants have effect on TF binding
• Does TF remain associated with ribosomes
  containing mutant L23?
• Can TF rebind ribosomes containing mutant L23?

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L23 Mutants and TF Binding

• SDS-PAGE of isolated ribosomes
• Control is from ?rplW cells with wt L23 from
  plasmid
• TF does not remain associated with mutant L23
  ribosomes

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L23 Mutants and TF Binding

• SDS-PAGE of ribosome-TF pellet and supernatant
• Control is from ?rplW cells with wt L23 from
  plasmid
• Little TF binds to mutant L23 ribosomes

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L23 Mutants and TF Binding

• Less TF co-purifies with ribosomes under
  physiological salt concentrations
• Mutant L23 levels are consistent with wt
  ribosomal proteins

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TF Interacts Directly with L23

• Create S-tagged L23-thioredoxin fusion (Trx-L23)
• Bind to S-tag column and apply TF or FRK/AAA
• Elute bound proteins

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TF Interacts Directly with L23

• TF binds L23, but FRK/AAA binding is weak
• TF and FRK/AAA have similar substrate binding
  properties
• L23-TF interaction is not mediated through
  nascent polypeptide

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TF Nascent Polypeptide Interaction and L23

• Must TF bind L23 to interact with nascent
  polypeptide?
• Use in vitro transcription/translation (IVT) and
  crosslinking
• Produce 35S-labeled isocitrate dehydrogenase
  (ICDH) fragment
• Use crosslinker to probe for TF-ICDH interaction

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In Vitro Transcription/ Translation System

• Translation competent fraction from ?tig E. coli
• Purified ribosomes with wt L23, region 1 L23
  mutants, or no L29
• Purified TF
• Produce N-terminal fragment of ICDH, an in vivo
  TF substrate

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Crosslinking

• Crosslinker is disuccinimidyl suberate (DSS)
• Homobifunctional
• Spans 11.4 angstroms
• Reacts with ?-amino groups of Lys to give
  crosslink and N-hydroxy succinimide (NHS)

DSS
NHS
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Identifying Crosslink Results

• Immunoprecipitate crosslink product with anti-TF
  Ab
• IP and non-IP samples examined by elecrophoresis,
  autoradiography
• Control with no DSS

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L23 is Required for TF ICDH Interaction

• wt L23 yields strong TF-ICDH crosslinks
• L23 mutants retard crosslinking
• Co-IP w/anti-TF Abs confirms identity
• Glu 18 mutants reduce TF-ICDH interaction

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TF-Ribosome Interaction and In Vivo Protein
Folding

• Combine ?rplWkan with ?dnaK
• Compensate with plasmids for wt or mutant L23
• Examine growth and aggregation at different
  temperatures

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TF-Ribosome Interaction and in vivo Protein
Folding

• wt L23 compensates for deletion
• L23 mutations lethal at 37ºC

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TF-Ribosome Interaction and in vivo Protein
Folding

• Aggregates isolated from double mutants
• Aggregation increases with temperature
• VSE/AAA mutation is most severe

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The Big Picture
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Conclusions

• L23 is the TF docking site on the ribosome
• Glu 18 is critical for binding
• Mutations in TF or L23 which inhibit binding
  affect protein folding, growth
• L23 couples protein synthesis with
  chaperone-assisted folding

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Future Directions

• Why does TF form two crosslinks to nascent
  chains?
• What is the nature of the L23-TF binding
  interface?
• Does temp increase rate of aggregation or TF-L23
  on-off rate?
• Role for eukaryotic L23 in recruiting chaperones?