1. Some examples of how NMR can provide information about biological systems

3. Autoinhibited Multi-Domain Proteins are Critical in Many Signal Transduction Pathways
Numerous multi-domain
proteins transmit signals
from the T-cell receptor
Need blocks to show the autoinhibtion.
Just to highlight the importance of autoinhibition, I am schematically describing a subset of interactions of a signal transduction pathway that originates at the cell surface receptor. This is the pathway from T cell receptor and it controls a number of important cellular events including reorganization of the actin cytoskeleton.
In this pathway, signal is triggered when extracellular ligands bind to the receptor, which in turn initiates a signaling cascade phosphorylation events of its intracellular domains.
I have colored in several molecules, this is by no means an exhaustive description of this signaling pathway, but the point is to highlight several proteins whose function is regulated through autoinhibition.
Lck, describe it
WASP
PAK1
And of course, I purposely left out Vav, which will be the topic of discussion today.
Rosen lab

4. Vav proto-oncoprotein is a key GEF that regulates Rho family GTPases
A member of the Dbl family of guanine nucleotide exchange factors (GEF) for the Rho family of GTP binding proteins.
Important in hematopoiesis, playing a role in T-cell and B-cell development and activation.
DH domain is inhibited by contacts with the Acidic (Ac) region and is relieved by phosphorylation of the Ac region tyrosines

5. A Helix From the Ac Domain Binds in the DH Active Site: Autoinhibition by Occlusion
This is a different view of the Y3-DH, which clearly show that the Y3 motif forms a helix.
As shown, the Y3 helix makes extensive contacts with the hydrophobic patch, which consists of xxx, yyy, and zzz residues.
One of the more interesting aspects of this structure is that the binding region of the Y3 helix also includes the predicted active site in the DH domain.
Therefore for the first time, we were able to rationalize the in vivo transforming data, specially the ones what showed that Y174 tyrosine in the Y3 motif, when mutate leads to transforming activity.
This study also looked at what happens to the activity and structure when the Y3 helix is phosphorylated.
Collectively, from this data the following model was described:
Y3 is buried in the interface
Aghazadeh, et al. Cell, 102:

6. Phosphorylation Disrupts Autoinhibitory Interactions
More succint use nmr spectrum
Swith 6 with 7
Aghazadeh, et al. Cell, 102:
Amide resonances from N-terminal (Ac region) helix collapse to the center of
the 1H/15N HSQC spectra and become extremely intense
13Ca and 13Cb assignments indicate that the N-terminus is random coil

7. How is Y3 Accessed by Kinases?
A general problem in autoinhibition/allostery: activators must contact buried sites ?

8. Chemical Shift Can Report on Population Distribution
Linearity of chemical
shifts across multiple
perturbations indicates
a two-state equilibrium
closed
50:50
mixture
We then design mutagenesis to find mutants with different open population. Based on the fact that the conformational changes are fast, the chemical shift of a residue undergoing conformational change is its population weighted position.
In the hypothetical case, closed state is at chemical shift 0 and open state is at 1. A mutant is 25% open, its chemical shift will be 0.25 according to this relationship. visa versa If the chemical shift of mutant is 0.25, its open population would be 25% according to the same relationship.
open
wobs = powo + (1-po)wc

9. Mutants Sample a Range of Population Distributions
Open
Closed
Use only the panel on the right, table w/ values and names for the mutants
W by omegas!!!!
Elaborate more on why we think these mutants are darm close to the sates in equlibirum
k208E!!!!!
Linearity strongly suggests an
equilibrium between Y3-
bound and Y3-unbound states
wobs = powo + (1-po)wc
Conformational equilibrium controls Vav activation by Src family kinases

17. Vav WASP/Cdc42 Rosen lab Science. 1998 Jan 23;279(5350):509-14.
Nature Mar 9;404(6774):151-8.
Nature May 27;399(6734):
Rosen lab