SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.

The cryptochrome (CRY) flavoproteins act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCF(FBXL3) ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitination and degradation. However, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound and FBXL3-SKP1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Notably, the F-box protein FBXL3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved carboxy-terminal tail and burying its PER-binding interface. This novel F-box-protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitination.

Site-specific incorporation of fluorotyrosines into proteins in Escherichia coli by photochemical disguise.

Fluorinated analogues of tyrosine can be used to manipulate the electronic environments of protein active sites. The ability to selectively mutate tyrosine residues to fluorotyrosines is limited, however, and can currently only be achieved through the total synthesis of proteins. As a general solution to this problem, we genetically encoded the unnatural amino acids o-nitrobenzyl-2-fluorotyrosine, -3-fluorotyrosine, and -2,6-difluorotyrosine in Escherichia coli. These amino acids are disguised from recognition by the endogenous protein biosynthetic machinery, effectively preventing global incorporation of fluorotyrosine into proteins.

Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity.

The type II secretion system (T2SS), a multiprotein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multidomain T2SS assembly ATPase GspE(EpsE) had not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspE(EpsE) are obtained when fused to Hcp1 as an assistant hexamer, as shown with native mass spectrometry. The enzymatic activity of the GspE(EpsE)-Hcp1 fusions is ∼20 times higher than that of a GspE(EpsE) monomer, indicating that increasing the local concentration of GspE(EpsE) by the fusion strategy was successful. Crystal structures of GspE(EpsE)-Hcp1 fusions with different linker lengths reveal regular and elongated hexamers of GspE(EpsE) with major differences in domain orientation within subunits, and in subunit assembly. SAXS studies on GspE(EpsE)-Hcp1 fusions suggest that even further variability in GspE(EpsE) hexamer architecture is likely.

Asymmetric synthesis of trisubstituted allenes: copper-catalyzed alkylation and arylation of propargylic phosphates.

Asymmetric synthesis of trisubstituted allenes is accomplished by copper-catalyzed alkylation and arylation of propargylic phosphates using organoboron nucleophiles. Excellent chirality transfer and regioselectivity, together with good functional group compatibility, were observed in reactions with both alkyl boranes and arylboronic esters.