The Molecular Basis of N End Rule Recognition Kevin H. Wang, Giselle - - PowerPoint PPT Presentation

The Molecular Basis of N‐End Rule Recognition

Kevin H. Wang, Giselle Roman‐Hernandez, Robert A. Grant, Robert T S d T i A B k

• T. Sauer, and Tania A. Baker

Molecular Cell 32, 406‐414, 2008 Speaker: Ching-Han Shen Advisor: Chii-Shen Yang, Ph.D.

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g

Protein degradation

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2004

Protein quality control Proteaosome q y Stress response

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Cell cycle regulation

prokaryotes eukaryotes

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N end rule in bacteria N‐end rule in bacteria

Bulky hydrophobic residues (Phe, Leu, Trp, Tyr)

How to establish binding specificity?

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ClpS cocrystal with N end rule peptide ClpS cocrystal with N‐end rule peptide

• C. crescentus ClpS 35‐119
• C. crescentus ClpS 35 119

Decapeptide with N‐terminal Tyr

M th d X diff ti

Method: X‐ray diffraction Resolution: 1.15 Å

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ClpS recognizes the first residue ClpS recognizes the first residue

Th bi di f f th N d l tid d th Cl A N d i

• The binding surfaces for the N‐end‐rule peptide and the ClpA N‐domain are

located on opposite sides

• Neither binding to a peptide substrate nor to the ClpA N‐domain appears to

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change the ClpS’s conformation substantially

• ClpS recognizes the first residue of N‐end rule substrates

α NH3+ pins the side chain in place α‐NH3 pins the side chain in place

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N-terminal peptide

Bulky hydrophobic side chain fits into the specificity pocket

a deep hydrophobic pocket

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a deep hydrophobic pocket

N-end rule residues: Phe, Leu, Trp, Tyr

Where does the specificity come from Where does the specificity come from

α‐NH3+ pins the side chain in place side chain fits into the specificity pocket side chain fits into the specificity pocket

To prove it further…

Conserved among evolution? What happened when mutated? What happened when mutated?

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ClpS contact residues are highly conserved ClpS contact residues are highly conserved

8 bacteria, 1 plant, 2 eukaryotic E3 ligase. S h l t d iti f N d l b t t

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Sequence homology suggests conserved recognition of N-end rule substrates.

Mutate Asn34 His66 Asp36 in E coli Mutate Asn34, His66, Asp36 in E. coli

Equivalent to Asn47 Asp49 His79 in Caulobacter Equivalent to Asn47, Asp49, His79 in Caulobacter

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Mutations cause defect in ClpAP degradation Mutations cause defect in ClpAP degradation

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Mutations compromise substrate recognition Mutations compromise substrate recognition

Michaelis-Menten plot

Vmax: how fast the enzyme can go at full speed

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• Vmax: how fast the enzyme can go at full speed
• Km: how much substrate is required to get to full speed.

β chain clashes sterically with Met53 β‐chain clashes sterically with Met53

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N-end rule residues: Phe, Leu, Trp, Tyr

Altered specificity mutation relieves restriction against Ile and Val

(~ M53 in Caulobactor)

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N-end rule residues: Phe, Leu, Trp, Tyr

Met53 acts as a specificity gatekeeper Met53 acts as a specificity gatekeeper

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N-end rule residues: Phe, Leu, Trp, Tyr

ClpS recognizes the first residue α-NH3+ pins the side chain in place Side chain fits into the specificity pocket p g

• f N-end rule substrates

Side chain fits into the specificity pocket How to explain the specificity?

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Contact residues are functionally important Met53 acts as a specificity gatekeeper

Thanks for your attention! y

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Proteolysis in eukaryotes Proteolysis in eukaryotes

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Proteolysis in prokaryotes Proteolysis in prokaryotes

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ClpAS specificity ClpAS specificity

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