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Biophysical Chemistry ILecture by Joachim Lätzer
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Overview
Introduction to phosphorylationExamples of different SignalsStructural ManifestationBiophysical characterization of phosphorylation
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Introduction to phosphorylation
Fischer and Krebs discoveryWhy phosphorylation is useful
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The Nobel Prize in Physiology or Medicine 1992
"for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism"
Edmond H. Fischer
Edwin G. Krebs
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Breakdown of glycogen to glucose1. Calcium signal2. Calcium activates
phosphorylase kinase3. Phosphorylase kinase
phosphorylatesphosphorylase, which is activated
4. Glycogen is broken to glucose ATP
5. Muscle work using ATP6. Muscle contains muscle
cells7. Contraction
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Reversible protein phosphorylation
PosttranslationalControl
Enzymatic reaction
ΔG~12kcal/mol
Kinasephosphorylates
Phosphatasedephospho-rylates
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Examples of different Signals
General ExamplesMAPK/ERK signaling pathway: cell divisionOncogene: Cancer and MAPK/ERKNF-KB signaling pathway: phosphorylation and inhibition
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General Examples
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MAPK/ERK Signaling Pathway
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Simplified view
1. Growth factor binds to a receptor
2. located in the cell membrane- this activates:
3. Grb24. Sos5. Ras6. Raf7. MEK8. Map Kinase
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What does map kinase (ERK) do?
Map kinase activates CDK1 which turns on cell divisionMap kinase enters the nucleus and activates transcription factorsTranscription factors then bind to DNA to turn on genes that lead to cell division
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Question: What happens if the signal pathway is modified ?
Usually cancer • Caused by viruses• Caused by mutations
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Example: Rous sarcoma virus (RSV)
gag - encodes capsid proteinspol - encodes reverse transcriptaseenv - encodes envelope proteinssrc - encodes a tyrosine kinase that attaches phosphate groups to the amino acid tyrosine in host cell proteins
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Example: Rous sarcoma virus (RSV)
v-src lacks the C-terminal inhibitory phosphorylation site (tyrosine-527), and is therefore constitutively active as opposed to normal src (c-src) Continuous cell profileration tumor
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MAPK/ERK and Cancer
“MAP kinase signalling pathways in cancer” Oncogene (2007) 26, 3279–3290
Inhibition through phosphorylation
Interference through phosphorylation
Modified cell communication CANCER
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Structural Manifestation
Conformational changeReorientationOrderingDisorderingAlternate Binding
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Structural Effect of Phosphorylation
Phosphorylation is an important regulatory mechanism
Can reversible attach/detach a phosphate and therefore switch “on”/”off” the function
Effect of phosphorylation is manifold• Conformational change• Ordering/disordering• Electrostatic effects• Alternate binding behavior
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Conformational Change in ERK2
Grey closed form
pTyr185 blocks binding site
Green open and active form
Johnson and Lewis, Chemical Reviews, 2001, Vol. 101, No. 8
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Reorientation: A conformational switch
Title: A phosphorylation-induced conformation change in dematin headpiece Author(s): Jiang ZHG, McKnight CJ Source: STRUCTURE Volume: 14 Issue: 2 Pages: 379-387 Published: FEB 2006
DHP (red) and DHPs74e (blue)
Rmsd = 2.5Å Z-Score = 4.6 (>3.6 same fold)
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Ordering events: KIX and pKID
KIX and phosphorylated pKID
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Disordering: NtrC, a molecular switch upon phosphorylation
Volkman et al., Science 2001, 291, 2429-33
Orange-yellow: unphosphorylated NtrCblue-cyan: phosphorylated NtrC
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Alternate Binding: SRC SH2 domain binding
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Phosphorylation leads to unbinding in the NF-KB signaling pathway
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Can we understand (predict) the effect of phosphorylation
ElectrostaticsHydrogen bondingSize
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Biophysical Methods
CrystallographyNMR methodsCryoelectronic imagingKinetic and thermodynamic measurementsModeling methods
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Early Wisdom on use of computations“There is a more general lesson to be drawn from the example of the genetic code. This is that, in biology, some problems are not suitable or not ripe for a theoretical attack for two broad reasons. The first I have already sketched--the current mechanisms may be partly the result of a historic accident. The other is that the “computations” involved may be exceedingly complicated.
“ . . . These difficulties do not mean we should not look for the broad principles involved (for example….), but it does mean that it may be better to try to go around such problems and not try to tackle them head on at too early a stage.” Francis Crick
“What Mad Pursuit: A Personal View of Scientific Discovery”Basic Books, 1988
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burialwatercontactRGAMBBAMW HHHHHHH +++++=
For HAM aligned to X-ray
Prediction of Phosphorylation Effects
Direct and water-mediated contacts, optimized with strategy based on maximizing TF/Tg (max Energy gap, min excess ruggedness )
Assure correct polypeptide chemistry
Hburial gives density preferences for each amino acid
Introduce 21st charge: supercharged glutamic acid to mimic phosphoresidue
AMW: Papoian et al., PNAS , 2004 ,101(10):3352-7.
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Prediction of pNtrC with the Phospho AMH
Rmsd = 2.3Å CE Z-Score = 6.0
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Example: SH2 domains as drug targets for diseases
Disease SH2 domain-containing target
AIDS Lck, HckAllergy and asthma Syk, LynAnaemia SHP-1Autoimmune disease ZAP-70Breast cancer Grb2, Grb7, SrcCancer p85, Shc, Grb2, GAP CML and ALL Grb2, CrklErythroleukaemia ShcInflammatory disease STATsPre-B-cell leukaemia BtkMyelodysplastic syndrome TecOsteoporosis Src
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Experimental data
Lubman, O.Y. and Waksman, G. , J Mol Biol 316 (2002) “Dissection of the energetic coupling across the Src SH2 domain‐tyrosyl phosphopeptide interface.”
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Analogues that bind SH2 domains
“Src Homology-2 Domains: Structure, Mechanisms, and Drug Discovery”, Sawyer, Biopolymers, 1998;47:243-63
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Binding Data
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Prediction with an AA force field
The AMBER force field
A sequence with unknown structure
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Results from MD simulationsUnphosphorylated
Phosphorylated
ArginineClaw
“A proposed signaling motif for nuclear import inmRNA processing via the formation of arginine claw”, Hamelberg et al, PNAS 2007, 104:14947-14951