How do proteins alter existing functions to form new ones?

Cameron BrownUniversity of Georgia

Mentor: Doug AndersonPrehoda LabSPUR 2011

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Anderson, 2010

Guanylate Kinase is a Model for Protein Evolution

Nucleotide kinase(GK enzyme) Protein-protein binding(GK domain)

+ GMP + ATP k cat+ GDP + ADP

GK enzyme

GK domain

How did this occur in evolution?

Anderson, 2010

evolution

GK Evolution Mechanism may be Applicable to other Metazoan Systems

GK domain

+ GMP + ATP

GK enzyme Enzyme

Protein binder

Acetylcholinesterase (AChE)

Neuroligins

Guanylate Kinase is a metabolic enzyme that phosphorylates GMP using ATP

Apo Gkenz

(pdb:1EX6)

GMP bound Gkenz

(pdb:1GKY)

+ GMP + ATP

GMP binding domain

Anderson, 2010

GK domains bind phosphoproteins without undergoing allosteric change

Apo GKdom

(pdb: 1JX0)

Pins bound GKdom

+ Phospho-Pins

Johnston, 2010

GK Domains diverged ~600 MYA from GK Enzymes

GK enz Vert. Dlg-1,4 Fly GKZo-likeMPP-like

GK Domains

Ancient GK enz

time

Ancestral Reconstruction allows us to go back in time…

GK enz Vert. Dlg-1,4 Fly GKZo-likeMPP-like

AncGK1

AncGK2

AncGK3

GK Domains

Ancestral Reconstruction

Ancient GK enz

time

AncGK0

…to uncover the path from ancient GK enzymes

to modern day GK domains

GK enz Vert. Dlg-1,4 Fly GKZo-likeMPP-like

AncGK1

AncGK2

AncGK3

GK Domains

Ancestral Reconstruction

Ancient GK enz

time

AncGK0

Question: Is AncGK1 an Enzyme, protein binder, or neither?

GK enz Vert. Dlg-1,4 Fly GKZo-likeMPP-like

GK Domains

Ancient GK enz

time

[SVSHTTR]

[CVPHTTR]

[SVSHTTR]AncGK1

AncGK0

AncGK2

AncGK3

Hypothesis: AncGK1 is an Enzyme

Analyzing Guanylate Kinase Catalytic Activity using Michaelis-Menten Kinetics

ATP + GMP ADP + GDPGK

ADP + GDP + 2PEP ATP + GTP + 2 pyruvatePK

2 Pyruvate + 2 NADH + 2H+ 2 Lactate + 2 NAD+LDH

Agarwal, 1978

Analyzing Guanylate Kinase Catalytic Activity using Michaelis-Menten Kinetics

ATP + GMP ADP + GDPGK

ADP + GDP + 2PEP ATP + GTP + 2 pyruvatePK

2 Pyruvate + 2 NADH + 2H+ 2 Lactate + 2 NAD+LDH

Agarwal, 1978

Analyzing Guanylate Kinase Catalytic Activity using Michaelis-Menten Kinetics

ATP + GMP ADP + GDPGK

ADP + GDP + 2PEP ATP + GTP + 2 pyruvatePK

2 Pyruvate + 2 NADH + 2H+ 2 Lactate + 2 NAD+LDH

Agarwal, 1978

Analyzing Guanylate Kinase Catalytic Activity using Michaelis-Menten

Kinetics

YGuk1WT:Vmax = 0.2086Km = 354.6 μM

AncGK1 is not a catalytically active GK enzyme

YGuk1WT:Vmax = 0.2086Km = 354.6 μM

Enzymatic activity was lost sometime after GK domains

diverged

GK enz Vert. Dlg-1-4 Fly GKZo-likeMPP-like

GK Domains

Ancient GK enz

time

AncGK1

AncGK0

Enzyme activity?

Future Directions

GK enz Vert. GK-1-4 Fly GKZo-likeMPP-like

AncGK1

AncGK2

AncGK3

GK Domains

Ancestral Reconstruction

Ancient GK enz

GKdom

time

AncGK0

AncGK0AncGK1Continue running towards GKdom

Acknowledgements • Prehoda Lab

– Ken– Doug– Chris– Matt– Marisa– Ryan– Oggie– Chiharu– Michelle– Jon– Brett– Amanda– Evan– Page

• SPUR– Peter– Lyndsey, Toby, and Blakely– The Interns