Some examples of how NMR can provide information about biological

systems

Autoinhibited Multi-Domain Proteins are Critical in Many Signal Transduction Pathways

• Numerous multi-domain proteins transmit signals from the T-cell receptor

Rosen lab

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

A Helix From the Ac Domain Binds in the DH Active Site: Autoinhibition by Occlusion

• Y3 is buried in the interfaceAghazadeh, et al. Cell, 102: 625-633.

Phosphorylation Disrupts Autoinhibitory Interactions

• 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

Aghazadeh, et al. Cell, 102: 625-633.

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

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Chemical Shift Can Report on Population Distribution

closed

open

50:50mixture

• Linearity of chemical shifts across multiple perturbations indicates a two-state equilibrium

wobs = powo + (1-po)wc

Mutants Sample a Range of Population Distributions

wobs = powo + (1-po)wc

Conformational equilibrium controls Vav activation by Src family kinases

Open

Closed

• Linearity strongly suggests an equilibrium between Y3- bound and Y3-unbound states

Rosen lab

Vav WASP/Cdc42

Nature. 1999 May 27;399(6734):379-83.

Nature. 2000 Mar 9;404(6774):151-8.

Science. 1998 Jan 23;279(5350):509-14.