Regulation of the Binding Function of PRDs

Although the binding affinities for PRDs are usually in the |M range, proteins containing these domains might nevertheless contribute critically to protein complex formation. Many of these complexes become activated when a certain biological stimulus arrives and complex assembly needs to be suppressed prior to activation. Therefore, activation-induced unmasking or compartmentalization of the PRD binding site is frequently observed in proteins. While certain WW domains have evolved into p-Ser-dependent modules (Lu et al. 1999), it has also been observed that post-translational modifications adjacent to the PRM affect the binding of SH3 domains in vitro (Bedford et al. 2000). The compartmentalization of PRD-contain-ing proteins will usually be assisted by domains other than PRDs, but it has also been observed that the PRD itself is absolutely required for the localization of certain proteins. Another possibility is that PRD:PRM interactions contribute to compartmentalization, but will be complemented or substituted by other, more specific interactions during the course of protein complex assembly. There are several examples of molecular complexes that rely on multiple domain interactions in conjunction with post-translational modifications. A well-described system is the NADPH oxidase, the catalytic and membrane-bound core of which consists of the gp91 and p22 subunits (Li 2005; Takeya et al. 2006). The cytosolic components p40, p47, p67 and Racl need to be recruited to the membrane for activation of the oxidase. A critical step in the assembly is the release of an autoinhibited conformation of the p47 protein. The two SH3 domains of this subunit are able to interact directly, forming a super-SH3 fold with a composite binding site that is masked by a basic C-terminal region comprising the motif RGAPPRRSS (Groemping et al. 2003). Upon stimulation of the cell, several serine residues in the polybasic region become phosphorylated, the auto-inhibited conformation is released, and binding of p47 to a classical PxxP motif within the membranous p22 subunit takes place. This example highlights several of the general mechanisms that are able to modulate the binding function of PRDs: (1) intramolecular interactions represent a means to prevent premature binding, (2) PRDs may form dimers with altered binding specificities, (3) sequences other than classical PxxP motifs are able to interact with the SH3 interaction site, and (4) post-translational modifications are able to couple indirectly to PRD scaffolding. Furthermore, the PX domain of p47 is also necessary for membrane recruitment of the protein, nicely exemplifying the additive or even cooperative binding of modular domains within the context of multiprotein complexes.

Another paradigmatic example of enzyme regulation by SH3 domains is the members of the Src family of tyrosine kinases. These proteins can exist in two major forms: an inactive, auto-inhibited conformation that is stabilized by intramolecular interactions between the SH2 domain and a phosphotyrosine motif and between the SH3 domain and a proline-helix-forming linker segment between the SH2 and kinase domain (Xu et al. 1999). Dephosphorylation of the inhibitory tyro-sine and intermolecular binding by the SH2 and SH3 domains leads to the formation of a fully active conformation that is able to phosphorylate several downstream targets. This "turned on by touch" behavior seems to be a remarkably common feature of tyrosine kinases in general, even in cases where binding partners are distinct and post-translational modifications differ (Boggon and Eck 2004). Targeting by SH3 domains and other adaptor domains is thereby unequivocally linked to the control of enzymatic activity.

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