Site directed mutagenesis to the rescue: unravelling conformational changes of Inositol 1,3,4,5,6 pentakisphosphate 2-kinase.

1 pa

A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation

© The Authors Journal compilation © 2014 Biochemical Society. IP3-3K [Ins(1,4,5)P33-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3is a key point in Ca2+metabolism that promotes Ca2+release from intracellular stores and together with Ins(1,3,4,5)P4regulates Ca2+homoeostasis. In addition, Ins(1,3,4,5)P4is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.Peer Reviewe

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage Φ29 DNA mimic protein p56.

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage 29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.Peer Reviewe