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Russell Group, Protein Evolution
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Putting it all together to answer a “real”
question
Rob Russell
Cell Networks
University of Heidelberg
Russell Group, Protein Evolution
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Domains assemble
to form higher-order
structures
Russell Group, Protein Evolution
Pawson & Nash, Science, 2003
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Case study 1: GabaB R1/R2
• Family 3 GPCRs
• Subunit R1 binds ligands, R2 signals,
but not vice versa
• Why?
Russell Group, Protein Evolution
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>gi|3776094|emb|CAA09940.1| GABAB receptor, subunit 1b [Homo sapiens]
MGPGAPFARVGWPLPLLVVMAAGVAPVWASHSPHLPRPHSRVPPHPSSERRAVYIGALFPMSGGWPGGQACQPAVEMALEDVNSRRDILPDYELKLIHHDSK
CDPGQATKYLYELLYNDPIKIILMPGCSSVSTLVAEARMWNLIVLSYGSSSPALSNRQRFPTFFRTHPSATLHNPTRVKLFEKWGWKKIATIQQTTEVFTSTLDDL
EERVKEAGIEITFRQSFFSDPAVPVKNLKRQDARIIVGLFYETEARKVFCEVYKERLFGKKYVWFLIGWYADNWFKIYDPSINCTVDEMTEAVEGHITTEIVMLNPA
NTRSISNMTSQEFVEKLTKRLKRHPEETGGFQEAPLAYDAIWALALALNKTSGGGGRSGVRLEDFNYNNQTITDQIYRAMNSSSFEGVSGHVVFDASGSRMAW
TLIEQLQGGSYKKIGYYDSTKDDLSWSKTDKWIGGSPPADQTLVIKTFRFLSQKLFISVSVLSSLGIVLAVVCLSFNIYNSHVRYIQNSQPNLNNLTAVGCSLALAA
VFPLGLDGYHIGRNQFPFVCQARLWLLGLGFSLGYGSMFTKIWWVHTVFTKKEEKKEWRKTLEPWKLYATVGLLVGMDVLTLAIWQIVDPLHRTIETFAKEEPK
EDIDVSILPQLEHCSSRKMNTWLGIFYGYKGLLLLLGIFLAYETKSVSTEKINDHRAVGMAIYNVAVLCLITAPVTMILSSQQDAAFAFASLAIVFSSYITLVVLFVPK
MRRLITRGEWQSEAQDTMKTGSSTNNNEEEKSRLLEKENRELEKIIAEKEERVSELRHQLQSRQQLRSRRHPPTPPEPSGGLPRGPPEPPDRLSCDGSRVHLL
YK
Analysis of intrinsic features
Signal peptide
Low complexity
PFAM analysis
Homology to known structure can be used to create
model
Russell Group, Protein Evolution
Transmembrane helices
Coiled coil region
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Family III GPCRs
Ligand binding domain
R1
cut
EC2
EC1
1
IC1
2
3
4
5
6
IC3
G-protein
7
dimerisation
IC2
---
Russell Group, Protein Evolution
EC3
Cterm
R1 binds ligand _________
R2 signals
Russell Group, Protein Evolution
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Robbins et al, J. Neurosci, 21, 8043, 2001
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GabaB R1/R2
Ligand binding domain
R1
EL2
EL1
(none)
EL1
EL3
1234567
IL1
IL2
- --
IL1
blocked
--G-protein
Russell Group, Protein Evolution
EL2
EL3
1234567
IL3
R2
Cterm
IL2
IL3
Cterm
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Case study 2: Human
RYK
an inactive tyrosine kinase
Russell Group, Protein Evolution
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Human RYK model
Insulin receptor YK
(template)
Human RYK
(model)
Russell Group, Protein Evolution Katso, Russell, Ganesan, Mol Cell Biol, 19, 6427, 1999
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Case study 3: What
are
phosphorylation sites doing?
Russell Group, Protein Evolution
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Van Noort et al, Mol Sys Biol, 2012
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MPN134 is phosphorylated
at Serine
392
Russell Group, Protein Evolution Katso, Russell, Ganesan, Mol Cell Biol, 19, 6427, 1999
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What do modifications do to interfaces?
From polar to negatively charged
From positively charged to polar
Modelled MPN134 homodimer:
From a polar-polar interaction to a pair
of negative charges in proximity
Russell Group, Protein Evolution
Van Noort et al, Mol Sys Biol, 2012
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Homology modelling
algorithm
+
Russell Group, Protein Evolution
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Homology modelling steps
•
•
•
Identify the homologue of known
structure
Get the best alignment of your
sequence to the structure
Model building
– Side-chain replacement
– Loop building
– Optimisation/relaxation/minimisation
Russell Group, Protein Evolution
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Russell Group, Protein Evolution
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Problem with loops
Two subtilisin-like serine proteases
Russell Group, Protein Evolution