Structure Watch

Two become three

Two domains are known to mediate protein–protein interactions by binding to phosphotyrosine (pTyr) residues in target proteins: Src-homology-2 (SH2) and pTyr-binding (PTB) domains. However, in Cell, Soltoff and colleagues have now discovered that a C2 domain (conserved domain 2) can bind to specific pTyr-containing peptides. This is an unusual finding, as these domains are known to mediate Ca²⁺-dependent lipid binding.

The authors focused on protein kinase Cδ (PKCδ), which contains a C2 domain that does not bind Ca²⁺ or phospholipids and which is regulated by the kinase Src. To understand this regulation, they first identified a protein that associates with both PKCδ and Src, CDCP1. They then showed that Tyr phosphorylation of CDCP1 correlates with an association between CDCP1 and PKCδ. This interaction was found to be mediated by the C2 domain of PKCδ binding to a pTyr residue in CDCP1, and a 1.7-Å-resolution crystal structure of the PKCδ C2 domain bound to an optimal phosphopeptide highlighted a new mode of pTyr binding. Although the phosphate is coordinated by an Arg residue, as in SH2 and PTB domains, Tyr binding involves a ring-stacking interaction with a His residue of the PKCδ C2 domain. PKCδ is the first example of a serine/threonine kinase that contains a pTyr-binding domain. Furthermore, data from this study indicate that Src phosphorylates and binds to CDCP1 through its SH2 domain, which promotes further CDCP1 phosphorylation and PKCδ recruitment to form a protein complex that contains two important kinases. REFERENCE Benes, C. H. et al. The C2 domain of PKCδ is a phosphotyrosine binding domain. Cell 121, 271–280 (2005)

Forced to let go

Nuclear transport is coordinated by nuclear Ran·GTP, which releases cargo from importin proteins and promotes cargo binding to exportin proteins. In Nature, Stewart and co-workers provide the basis for understanding the important cargo-release step of nuclear import by presenting the 2.7-Å-resolution crystal structure of full-length yeast importin-β, Kap95, bound to Ran·GTP.

Kap95 is composed of two C-shaped arches that are linked together to form a flexible helicoidal stucture. A previous structure that included only the N-terminal arch of importin-β highlighted two important interaction sites with Ran, one of which involved the switch II region (the conformations of the Ran switch I and II regions differ between the GTP- and GDP-bound states). However, this structure containing full-length Kap95 highlights the importance of a third interaction site between the C-terminal arch of Kap95 and the switch I region of Ran·GTP. By exploiting its flexibility and by using different binding sites, importin-β/Kap95 can bind to a range of different cargoes. So, how does Ran·GTP binding release all of these cargoes? On the basis of their structure, the authors propose that, by binding to sites in both the C- and N-terminal arches, Ran·GTP actively displaces cargo and locks importin-β/Kap95 “...in a conformation in which it is unable to exploit its flexibility to bind to different partners”. REFERENCELee, S. J. et al. Structural basis for nuclear import complex dissociation by RanGTP. Nature 1 May 2005 (10.1038/nature03578)