Abstract
T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PHn-PHc), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.
Highlights
We present a series of novel findings regarding the mechanism and structural basis of Tiam1 GEF auto-inhibition
Our results establish a novel mechanism for autoinhibition of Tiam1 by intramolecular interactions through the PHn-CC-Ex domain
We determined the enzyme kinetics of Tiam1-catalyzed nucleotide exchange reactions on Rac1 and found that inhibition occurs by a competitive model where the auto-inhibition is due to a lower substrate-binding affinity rather than inhibition of catalysis
Summary
To investigate the role of structured domains in regulating GEF catalytic activity, we constructed a series of fragments with sequentially deleted domains (Fig. 1A). The trueKm value of the PHn-PHc fragment was ϳ2.4-fold higher than that of the DH-PHc fragment These data indicate that the inhibition of Tiam GEF function occurs through a competitive model whereby the PHC-CC-Ex and RBD domains inhibit the DH-PHc domain by decreasing Rac1-binding affinity. The Kratky analysis of the scattering data indicated that all Tiam proteins had folded structure, but the increase of the curve at high q range suggests that they have regions of flexibility, the PHn-PHc fragment (Fig. 5D). To understand how intramolecular interactions suppress the GEF activity of the Tiam PHn-PHc fragment, we created SAXS-based structural models using ab initio and rigid body approaches. Control experiments indicated that the biotin label had no effect on GEF exchange activity of the PHn-PHc fragment and a small effect on the DHPHc domain (supplemental Fig. S8). Frequent events of Rac diffusion through the diffraction-limited spot around the surface-tethered PHn-PHc (or DH-PHc) prevented reliable assignment of binding events
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
