Protein Tyrosine Phosphatase 1B

1
Protein Tyrosine Phosphatase 1B

• Lauren Buerkel and Ed Lane
• May 2, 2006
• Overview
• The Protein Tyrosine Phosphatase (PTP) Family
• PTP1B Structure and Specificity
• Dephosphorylation of pTyr by PTP1B
• Inhibition of PTP1B by a Bidentate Inhibitor

2
The PTP Family

• Important role in cellular signaling, either
  positively or negatively
• Such as Normal Cell Growth, Hormones, Cell Cycle,
  Immune Response, etc.
• Work with the Protein Tyrosine Kinase (PTK)
  family to regulate protein functions
• Dephosphorylation by PTPs and phosphorylations by
  PTKs
• Tyrosine Specific or Dual Specific
• Dual Specific PTPs have the ability to
  dephosphorylate pTyr and pSer/PThr

3
PTP1B

• A negative regulator of insulin and leptin
  pathway signaling
• Tyrosine Specific PTP with a single catalytic
  domain
• 8 alpha helices, 12 beta sheets
• 3 Important Loops
• Catalytic or PTP
• Cys215 and Arg221
• Recognition
• Substrate Recognition
• Val49 and Tyr46
• WPD
• Transforms to a closed configuration once
  dephosphorylation occurs

Red N- and C- Termini Yellow Catalytic
Site Blue Loop Sequences Light Blue- Invariant
Residues
4
PTP1B Catalytic Site

• H-bonds orientate substrate to the catalytic loop
• Beta-5 and Beta-6
• Arg257 A buried charged residue
• Stabilized by H-bonds with Asn68, Tyr66, Asn44
• Increase electrostatic potential and binding
  affinity

5
Specificity of PTP1B

• The Recognition Pocket
• Base Asn221 Rim Asp48
• Deep Specific to pTyr
• Forms H-bonds and saltbridges with 3 terminal
  phosphate oxygens on pTyr to stabilize
• Hydrophobic interactions between non polar side
  chains lining catalytic site and phenyl ring of
  pTyr

6
Dephosphorylation of pTyr

• A pTyr residue enters the catalytic site
• The WPD loop transforms into the closed
  confirmation stabilizing the substrate into
  position within the active site.
• Cys215 nucleophilically attacks the phosphate
  group to form intermediate pCys
• Water breaks the p-Cys bond, and the enzyme
  returns to standard confirmation

7
PTP1B Inhibition

• Why do we want a PTP1B inhibitor?
• Negative insulin and leptin regulator
• Enhance selectivity to treat type II diabetes and
  obesity
• Problems?
• pTyr active site is highly conserved in PTPs
• PTP1B plays a role in many signaling pathways and
  inhibiting may give undesirable side effects
• Specificity to PTP1B
• Discovery of an adjacent phosphate binding site
  to the catalytic site
• Not conserved
• Bidentate inhibitor

8
Bidentate Inhibitor

• Compounds were purified and crystallized with
  PTP1B
• Hanging drop or sitting drop vapor diffusion
  method
• Determined structure of PTP1B/inhibitor
• Comparing Compound 1 and 2
• Similar potency
• Addition of Jeffamine 600 additive improved
  Compound 2 crystals

9
PTP1B/Compound 2

• Similar to PTP1B/pTyr
• WPD closed
• F2Pmp in active site
• 3 phosphate oxygens form H-bonds with PTP loop
• Phenyl rings interact with Tyr46 Phe182
• Unique to PTP1B/2
• Two fluorine atoms replace phenolic oxygen
• H-bonds with H2O
• Van der Waals with side chains
• Increases affinity
• Arg47
• Position allows interactions with both Asp linker
  and distal aryl difluorophosphonate group
• Asp48, Arg47, Lys41
• Adjacent to active site
• Form interactions that increase affinity

PTP1B/Compound 2
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PTP1B/2 Interactions

• Extensive interactions between side chains and
  Compound 2 increase affinity compared to pTyr
• H-bonding and hydrophobic interactions between
  Arg47, Asp48, Lys41, and compound 2 increase
  interaction
• Two fluorine atoms interaction with a water
  molecule
• Blue H-bonding
• Red Polar Interactions
• Orange Hydrophobic Interactions

11
PTP1B in Insulin Pathway

• Homo sapiens pathway
• PTPN1

12
Problems

• The difluorophosphonate portion of the bidentate
  inhibitors effect the proteins ability to cross
  cell membranes
• Some difluorophosphonate inhibitors imitate
  insulin activity