1

“L23 Protein Functions as a Chaperone Docking Site

on the Ribosome”Kramer, G., et. al. (2002)

Nature 419 171-174

Presented by Michael EvansDepartment of Chemistry and BiochemistryUniversity of Notre DameNotre Dame, IN 46616

2

Overview

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

3

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

4

Chaperone Pathway in Bacteria

Hartl, F.U. and Hayer-Hartl, M. (2002) Science 295 1852-1858

5

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

6

Significance

• Explain coupling of synthesis to folding

• Eukaryotic parallels– No TF– Other chaperones interact with

ribosome– SRP study

7

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 TF’s participation in protein folding?

8

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

9

TF Signature and Mutants

10

TF Signature Mutants

• FRK/AAA: should show reduced association with ribosomes

11

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)

12

FRK/AAA Mutant Association with Ribosomes

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

S: SupernatantP: Ribosome Pellet

13

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

14

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

15

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

16

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

17

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?

18

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

19

L23 and L29 Deletion Mutants

• Two mutants produced: rpmC::kan, deletion of L29 gene rplW::kan, deletion of L23 gene

rpmC::kan grows, but slightly slower than wt

rplW::kan requires presence of pL23 for growth

20

L23 and L29 Deletion Mutants

rplW::kan growth dependent on IPTG induction of pL23•L23 mutant is also viable

21

L29 and TF Binding

• Purify ribosomes from rpmC::kan under high salt conditions

• Does TF remain bound to ribosomes without L29?

• Can TF rebind ribosomes without L29?

22

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

23

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

24

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?

25

L23 Region 1 and 2 Mutants• Criteria for interaction:

– residue is surface-exposed– Conserved among bacterial L23s

• Two regions identified

26

L23 Region 1 and 2 Mutants

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

27

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?

28

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

29

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

30

L23 Mutants and TF Binding

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

31

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

32

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

33

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

34

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

35

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

36

Identifying Crosslink Results

• Immunoprecipitate crosslink product with anti-TF Ab

• IP and non-IP samples examined by elecrophoresis, autoradiography

• Control with no DSS

37

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

38

TF-Ribosome Interaction and In Vivo Protein Folding

• Combine rplW::kan with dnaK• Compensate with plasmids for wt

or mutant L23• Examine growth and aggregation

at different temperatures

39

TF-Ribosome Interaction and in vivo Protein Folding

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

40

TF-Ribosome Interaction and in vivo Protein Folding

•Aggregates isolated from double mutants

•Aggregation increases with temperature

•VSE/AAA mutation is most severe

41

The Big Picture

42

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

43

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?