A complex interaction

The cyclin-dependent kinase inhibitor Sic1 must be destroyed to allow cells to progress through the cell cycle, and this destruction occurs when Sic1 is phosphorylated on at least six of its nine Cdc4-phosphodegron (CPD) sites. This phosphorylated Sic1 is bound by the WD40 domain of the F-box protein Cdc4, which takes Sic1 to the SCFCdc4 (Skp1/Cullin/F-box protein) ubiquitin-ligase complex — a multisubunit E3 enzyme. Here, Sic1 is ubiquitylated, which targets it for proteolytic destruction. To clarify the basis of phospho-epitope recognition by Cdc4, and to further understand how E3 enzymes orientate their substrates, Sicheri and colleagues now describe in Cell the 2.7-Å X-ray crystal structure of a Skp1–Cdc4 complex bound to a CPD phosphopeptide.

The structure showed that a core CPD motif — Leu-Leu-pThr-Pro — binds to an eight-bladed β-propeller WD40 domain in Cdc4, and it also clarified how F-box proteins present substrates for ubiquitin transfer. Furthermore, the authors found that the low-affinity binding of CPD motifs in Sic1 to Cdc4 reflects a structural incompatibility with the CPD-binding site in Cdc4. When they re-engineered Cdc4 to optimize Sic1 binding, lower phosphorylated forms of Sic1 were ubiquitylated. These data explain the phosphorylation threshold for Sic1 binding, and indicate “...an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1”. REFERENCE Orlicky, S. et al. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase. Cell 112, 243–256 (2003)

An active Holliday

During genetic recombination, two homologous DNA molecules exchange strands to form a four-way DNA (Holliday) junction, and the subsequent action of junction-resolving enzymes determines the final genetic outcome. In the absence of Mg2+, the centre of this junction is unstacked and open, and the four helical arms point towards the corners of a square. However, in the presence Mg2+, the junction folds to form one of two possible stacked X-structures, in which two DNA strands run straight through a pair of stacked helices and the other two are swapped between helical pairs. It is thought that transitions occur between these alternative stacking conformers, but no direct evidence has been presented to support this idea. Now, though, in Nature Structural Biology, Ha and colleagues describe the use of single-molecule methodology to detect these transitions in real time.

The authors found that the processes of conformer transition and branch migration both have the unstacked, open structure as the common intermediate, but that conformer transitions occur much faster than branch migration steps. Correlations have been observed between the dominant stacking conformation and the resolution of a Holliday junction, which indicates that DNA sequences can affect the outcome of genetic recombination by biasing the stacking conformation. The results of this study therefore indicate that “...sequence-dependent conformer bias can be fully manifested even in a fully branch-migratable junction and can determine the extent of genetic information exchange upon junction resolution”. REFERENCE McKinney, S. A. et al. Structural dynamics of individual Holliday junctions. Nature Struct. Biol. 10, 93–97 (2003)