Russell Group, Protein Evolution

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Russell Group, Protein Evolution

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Rob RussellCell Networks

University of Heidelberg

Putting it all together to answer a “real” question

Russell Group, Protein Evolution

_________ ____Domains assemble to form higher-order structures

Pawson & Nash, Science, 2003

Russell Group, Protein Evolution

_________ ____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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Analysis of intrinsic features

Low complexity

Signal peptide

Coiled coil region

Transmembrane helices

>gi|3776094|emb|CAA09940.1| GABAB receptor, subunit 1b [Homo sapiens]

MGPGAPFARVGWPLPLLVVMAAGVAPVWASHSPHLPRPHSRVPPHPSSERRAVYIGALFPMSGGWPGGQACQPAVEMALEDVNSRRDILPDYELKLIHHDSKCDPGQATKYLYELLYNDPIKIILMPGCSSVSTLVAEARMWNLIVLSYGSSSPALSNRQRFPTFFRTHPSATLHNPTRVKLFEKWGWKKIATIQQTTEVFTSTLDDLEERVKEAGIEITFRQSFFSDPAVPVKNLKRQDARIIVGLFYETEARKVFCEVYKERLFGKKYVWFLIGWYADNWFKIYDPSINCTVDEMTEAVEGHITTEIVMLNPANTRSISNMTSQEFVEKLTKRLKRHPEETGGFQEAPLAYDAIWALALALNKTSGGGGRSGVRLEDFNYNNQTITDQIYRAMNSSSFEGVSGHVVFDASGSRMAWTLIEQLQGGSYKKIGYYDSTKDDLSWSKTDKWIGGSPPADQTLVIKTFRFLSQKLFISVSVLSSLGIVLAVVCLSFNIYNSHVRYIQNSQPNLNNLTAVGCSLALAAVFPLGLDGYHIGRNQFPFVCQARLWLLGLGFSLGYGSMFTKIWWVHTVFTKKEEKKEWRKTLEPWKLYATVGLLVGMDVLTLAIWQIVDPLHRTIETFAKEEPKEDIDVSILPQLEHCSSRKMNTWLGIFYGYKGLLLLLGIFLAYETKSVSTEKINDHRAVGMAIYNVAVLCLITAPVTMILSSQQDAAFAFASLAIVFSSYITLVVLFVPKMRRLITRGEWQSEAQDTMKTGSSTNNNEEEKSRLLEKENRELEKIIAEKEERVSELRHQLQSRQQLRSRRHPPTPPEPSGGLPRGPPEPPDRLSCDGSRVHLLYK

PFAM analysis

Homology to known structure can be used to create model

Russell Group, Protein Evolution

_________ ____Family III GPCRs

1 2 3 4 5 6 7

R1

Ligand binding domain

IC1 IC2IC3

Cterm

EC1 EC2 EC3

-- -

G-protein

dimerisation

cut

Russell Group, Protein Evolution

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Robbins et al, J. Neurosci, 21, 8043, 2001

R1 binds ligandR2 signals

Russell Group, Protein Evolution

_________ ____GabaB R1/R2

1 2 3 4 5 6 7 1 2 3 4 5 6 7

R1 R2

Ligand binding domain

IL1 IL2

IL3 Cterm

EL1 EL2 EL3

IL1 IL2IL3 Cterm

EL1 EL2 EL3

--

- -

-- -

G-protein

(none)

blocked

Russell Group, Protein Evolution

_________ ____Case study 2: Human RYKan inactive tyrosine kinase

Russell Group, Protein Evolution

_________ ____Human RYK model

Insulin receptor YK(template)

Human RYK(model)

Katso, Russell, Ganesan, Mol Cell Biol, 19, 6427, 1999

Russell Group, Protein Evolution

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Van Noort et al, Mol Sys Biol, 2012

Case study 3: What are phosphorylation sites doing?

Russell Group, Protein Evolution

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Katso, Russell, Ganesan, Mol Cell Biol, 19, 6427, 1999

MPN134 is phosphorylated at Serine 392

Russell Group, Protein Evolution

_________ ____What do modifications do to interfaces?

Van Noort et al, Mol Sys Biol, 2012

From positively charged to polar

From polar to negatively charged

Modelled MPN134 homodimer: From a polar-polar interaction to a pair of negative charges in proximity

Russell Group, Protein Evolution

_________ ____Homology modelling

+

algorithm

Russell Group, Protein Evolution

_________ ____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

_________ ____Problem with loops

Two subtilisin-like serine proteases