principles of protein phosphorylation - casegroup
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Principles of protein phosphorylationBiophysical Chemistry 1, Fall 2010
Signalling “cascades”Structural biology of phosphorylation
Web assignment: http://pkr.genomics.purdue.edu
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Kinases and phosphatases
Reversible protein phosphorylation
PosttranslationalControl
Enzymatic reaction
ΔG~12kcal/mol
Kinasephosphorylates
Phosphatasedephospho-rylates
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Signalling overviewGeneral Examples
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Cells are way too complex!MAPK/ERK Signaling Pathway
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Rous sarcoma virus (RSV)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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Mutations, viruses and cancerExample: 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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Biophysics of signallingStructural 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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Signalling by reorientationReorientation: 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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Order/disorder transitionsDisordering: 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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Src/SH2 interactions: binding vs releaseAlternate Binding: SRC SH2 domain binding
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Expected conformational effectsCan we understand (predict) the effect of phosphorylation
ElectrostaticsHydrogen bondingSize
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Mutational analysis
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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Looking for analoguesAnalogues that bind SH2 domains
“Src Homology-2 Domains: Structure, Mechanisms, and Drug Discovery”, Sawyer, Biopolymers, 1998;47:243-63
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Expected conformational effectsBinding Data
“Src Homology-2 Domains: Structure, Mechanisms, and DrugDiscovery”, Sawyer, Biopolymers, 47, 243-63 (1998)