A critical step involved in regulating the cytoskeleton and cell signalling is the activation of Rho guanosine triphosphatases (GTPases), initiated by the exchange of guanine diphosphate (GDP) for guanosine triphosphate (GTP), a process catalysed by guanine nucleotide exchange factors (GEFs). Here structural research provides us with evidence that a nucleotide sensor on the GEF DOCK9 is intrinsic to the release of guanine diphosphate (GDP) from the Rho-family GTPase Cdc42. This nucleotide sensor, comprising a conserved valine residue, is located on the α10 helix of the catalytic DHR2 domain of DOCK9. It functions by displacing magnesium bound to GDP, thereby weakening GDP affinity for Cdc42. However, tight binding of Mg2+ to GTP bound to Cdc42 disorders the nucleotide sensor, promoting discharge of activated Cdc42-GTP from DOCK9. This research was published in the journal Science.
Rho guanosine triphosphatases (GTPases) are activated by the exchange of guanine diphosphate (GDP) for guanosine triphosphate (GTP). This is a critical and necessary step involved in cell motility, adhesion, apoptosis and proliferation. Activators of this process include two distinct families of the Rho guanine nucleotide exchange factors (GEFs), the Dbl homology (DH) and DOCK proteins. DOCK proteins have been specifically implicated in the activation of Rac and Cdc42 and in the regulation of migration, phagocytosis and morphogenesis. DOCK proteins have also been implicated in tumour cell movement and invasion. All have a GEF catalytic domain called DHR2. At present the actual mechanism by which DOCK proteins activates GTPases is not known.
Here the structure of DOCK9 DHR2 domain was analysed, solved in complex with nucleotide-free Cdc42 and in complex with GDP-Cdc42 and GTP-Cdc42 to enable a complete view of the GEF catalytic cycle. Below is the solved structure of the DOCK9DHR2-Cdc42.
|Figure 1:The catalytic domain of GEF forming a stable complex with nucleotide-free GTPase (A) Lobes A,B and C are blue, cyan and green respectively. Cdc42 is orange with switch 1 and 2 and P loop in red, purple and blue respectively. α10 insert of DHR2 domain, with Val1951 is yellow. (B) Switch 1 dominates interactions between DOCK9DHR2 and Cdc42. Image from Science.|
Notably, according to the solved structure, the catalytic domain of the GEF is completely different from any other GEF catalytic domain.
The DOCK9DHR2-Cdc42-GDP structure was also solved to enable an understanding of how DOCK9 reduces Cdc42’s affinity for the nucleotide. The structural analysis indicated no protein conformational change in comparison to the complex of DOCK9 bound to nucleotide free Cdc42. When bound to DOCK9, switch 1 of Cdc42 alters its conformation and exposes the Cdc42’s nucleotide-binding site (fig 2A). The movement of switch 1 was linked to a rotation of the P-loop residue Cys18 (fig 2A). The conformational changes of switch 1 are linked to the intrusion into the GTPase nucleotide-binding site, of Val1951 of the α10 insert, thereby occluding the nucleotide-coordinated Mg2+(figure 2B). Since magnesium enhances nucleotide affinity, its exclusion profoundly decreases nucleotide affinity. Magnesium exclusion within the catalytic domain was found to be very different from any other GEF where the displacement of switch 2 Ala residue interfered with Mg2+ binding.
|Comparison of DOCK9DHR2-Cdc42 structures with isolated Cdc42-nucleotide complexe. Image from Science.|
The structure for the GTP bound complex was also solved (figure 2C). Association of Mg2+ after GTP binding is incompatible with the closed conformation of Val1951 of α10. Thus, displacement of the α10 insert removes the clamp from switch 1 and induces conformational changes of switch 1 of the GTPase towards the activated state, facilitating discharge of Cdc42-GTP from DOCK9, hence completing the catalytic cycle.
“The structure of DOCK9DHR2 in complex with Cdc42 provides a molecular explanation for how this unconventional family of GEFs mediates activation of Rho GTPases. This will provide a framework for structure-based drug design efforts to develop inhibitors of DOCK proteins that block activation of Rho GTPases, and hence cell migration that underlies cancer metastasis.”
Dr David Barford, Institute of Cancer Research
Activation of Rho GTPases by DOCK Exchange Factors Is Mediated by a Nucleotide Sensor, Jing Yang, Ziguo Zhang, S. Mark Roe, Christopher J. Marshall, David Barford, Science 11 (325) pp. 1398 - 1402, September 2009
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