To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys ⁴⁸ -linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.

The solution structure of the copper‐free state of a monomeric form of superoxide dismutase (153 amino acids) was determined through ¹³C and ¹⁵N labeling. The protein contained two mutations at the native subunit–subunit interface (F50E and G51E) to obtain a soluble monomeric species and a mutation in the active site channel (E133Q). About 93% of carbon atoms, 95% of nitrogen atoms, and 96% of the protons were assigned. A total of 2467 meaningful NOEs and 170 dihedral angles provided a family of 35 conformers with RMSD values of 0.76 ± 0.09 Å for the backbone and 1.22 ± 0.13 Å for all heavy atoms. The secondary structure elements, connected by loops, produce the typical superoxide dismutase Greek key fold, formed by an eight‐stranded β‐barrel. The comparison with the copper‐bound monomeric and dimeric structures shows that the metal ligands have a conformation very close to that of the copper‐bound forms. This feature indicates that the copper‐binding site is preorganized and well ordered also in the absence of the copper ion. The active‐site channel shows a sizable increase in width, achieving a suitable conformation to receive the copper ion. The histidines ring NH resonances that bind the copper ion and the region around the active‐site channel experience, as found from ¹⁵N relaxation studies, conformational exchange processes. The increased width of the channel and the higher mobility of the histidine rings of the copper site in the copper‐free form with respect to the holoprotein is discussed in terms of the process of copper insertion.

Calmodulin-like (CML) proteins are major EF-hand–containing, calcium (Ca²⁺)–binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca²⁺ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana. We analyzed Ca²⁺ and Mg²⁺ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca²⁺/Mg²⁺ mixed binding sites and two low-affinity Ca²⁺-specific sites. Binding of Ca²⁺ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca²⁺ or Mg²⁺, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca²⁺-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca²⁺ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca²⁺ pumps.