Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms

National Institute of General Medical Sciences R01 GM0880555T32 GM007270, Francis Crick Institute FCI01, Cancer Research UK, Medical Research Council U117565398, Wellcome Trus

Following Ariadne's thread: a new perspective on RBR ubiquitin ligases

Ubiquitin signaling pathways rely on E3 ligases for effecting the final transfer of ubiquitin from E2 ubiquitin conjugating enzymes to a protein target. Here we re-evaluate the hybrid RING/HECT mechanism used by the E3 family RING-between-RINGs (RBRs) to transfer ubiquitin to substrates. We place RBRs into the context of current knowledge of HECT and RING E3s. Although not as abundant as the other types of E3s (there are only slightly more than a dozen RBR E3s in the human genome), RBRs are conserved in all eukaryotes and play important roles in biology. Re-evaluation of RBR ligases as RING/HECT E3s provokes new questions and challenges the field

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein

The solution structure of the histidine-containing protein (HPr) from Staphylococcus aureus as determined by two-dimensional ¹H-NMR spectroscopy

The three-dimensional solution structure of the heat-stable phosphocarrier protein HPr from Staphylococcus aureus was determined from two-dimensional NMR data by restrained molecular dynamics. It consists of a large twisted antiparallel beta-pleated sheet with four strands A, B, C, and D of amino acids 2-7, 34-37, 40-42 and 60-65. Three right-handed helices A, B, C (amino acids 18-27, 47-53 and 71-85) are positioned on top of this sheet. The aromatic ring of His15 is located in a cleft formed by amino acids 12-17 and 55-58, only the nitrogen (N delta 1) atom which can be phosphorylated by enzyme I is exposed to the water. The side chains of Thr12 and Arg17 are located close to the histidine ring. The regulatory serine residue (Ser46) is located in a hydrophobic patch, its hydroxyl group is water-accessible but forms hydrogen bonds with the amide groups of the backbone. The general features of the three-dimensional structure are similar to those found in HPr proteins from different microorganisms such as Escherichia coli, Bacillus subtilis and Streptococcus faecalis