The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/β-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.

Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible ''pocket'' domain loop such that it mimics and directly blocks E2F transactivation domain (E2F TD ) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2F TD -pocket association and protein binding to the pocket ''LxCxE'' site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

Inactivation of the retinoblastoma protein (Rb) through phosphorylation is an important step in promoting cell cycle progression, and hyperphosphorylated Rb is commonly found in tumors. Rb phosphorylation prevents its association with the E2F transcription factor; however, the molecular basis for complex inhibition has not been established. We identify here the key phosphorylation events and conformational changes that occur in Rb to inhibit the specific association between the E2F transactivation domain (E2F TD ) and the Rb pocket domain. TD and the Rb pocket and describe for the first time a function for the structured N-terminal domain in Rb inactivation.

The emergence of catalysis in a noncatalytic protein scaffold is a rare, unexplored event. Chalcone isomerase (CHI), a key enzyme in plant flavonoid biosynthesis, is presumed to have evolved from a nonenzymatic ancestor related to the widely distributed fatty-acid binding proteins (FAPs) and a plant protein family with no isomerase activity (CHILs). Ancestral inference supported the evolution of CHI from a protein lacking isomerase activity. Further, we identified four alternative founder mutations, i.e., mutations that individually instated activity, including a mutation that is not phylogenetically traceable. Despite strong epistasis in other cases of protein evolution, CHI's laboratory reconstructed mutational trajectory shows weak epistasis. Thus, enantioselective CHI activity could readily emerge despite a catalytically inactive starting point. Accordingly, X-ray crystallography, NMR, and molecular dynamics simulations reveal reshaping of the active site toward a productive substrate-binding mode and repositioning of the catalytic arginine that was inherited from the ancestral fatty-acid binding proteins.