Binding stoichiometry and structural model of the HIV-1 Rev/Importin beta complex

HIV-1 Rev mediates the nuclear export of intron-containing viral RNA transcripts and is essential for viral replication. Rev is imported into the nucleus by the host protein Importin beta (ImpB), but how Rev associates with ImpB is poorly understood. Here we report biochemical, biophysical and structural studies of the ImpB/Rev complex. Binding and mutagenesis data reveal that ImpB associates with two Rev monomers through independent binding sites and that the N-terminal half of Rev's Arginine-Rich Motif (ARM) is a primary ImpB binding epitope. Small-angle X-ray scattering (SAXS), crosslinking mass spectrometry and compensatory mutagenesis data suggest a structural model in which one Rev monomer binds to the C-terminal half of ImpB with Rev helix alpha-2 roughly parallel to the HEAT-repeat superhelical axis while the other monomer binds to the N-terminal half. These findings shed light on the molecular basis of Rev recognition by ImpB and highlight an atypical binding behaviour that distinguishes Rev from canonical cellular ImpB cargos.

Copper(II) Complexes of N-Propargyl Cyclam Ligands Reveal a Range of Coordination Modes and Colours, and Unexpected Reactivity

<p>Herein we describe single crystal X-ray diffraction and spectroscopic investigations of the coordination chemistry of copper(II) complexes of cyclam derivatives with between 1 and 4 pendant alkynes. The crystal structures of these copper complexes unexpectedly reveal a range of coordination modes, and the surprising occurrence of five unique complexes within a single recrystallisation of the tetra-<i>N</i>-propargyl cyclam ligand. One of these species exhibits weak intramolecular copper-alkyne coordination, and another is formed by a surprising intramolecular copper-mediated hydroalkoxylation reaction with the solvent methanol, transforming one of the pendant alkynes to an enol ether. Multiple functionalisation of the tetra-<i>N</i>-propargyl ligand is demonstrated <i>via</i> a ‘tetra-click’ reaction with benzyl azide, and the copper-binding behaviour of the resulting tetra-triazole ligand is characterised spectroscopically.</p>

Copper(II) Complexes of N-Propargyl Cyclam Ligands Reveal a Range of Coordination Modes and Colours, and Unexpected Reactivity

<p>Herein we describe single crystal X-ray diffraction and spectroscopic investigations of the coordination chemistry of copper(II) complexes of cyclam derivatives with between 1 and 4 pendant alkynes. The crystal structures of these copper complexes unexpectedly reveal a range of coordination modes, and the surprising occurrence of five unique complexes within a single recrystallisation of the tetra-<i>N</i>-propargyl cyclam ligand. One of these species exhibits weak intramolecular copper-alkyne coordination, and another is formed by a surprising intramolecular copper-mediated hydroalkoxylation reaction with the solvent methanol, transforming one of the pendant alkynes to an enol ether. Multiple functionalisation of the tetra-<i>N</i>-propargyl ligand is demonstrated <i>via</i> a ‘tetra-click’ reaction with benzyl azide, and the copper-binding behaviour of the resulting tetra-triazole ligand is characterised spectroscopically.</p>

Alternative splicing and allosteric regulation modulate the chromatin binding of UHRF1

UHRF1 is an important epigenetic regulator associated with apoptosis and tumour development. It is a multidomain protein that integrates readout of different histone modification states and DNA methylation with enzymatic histone ubiquitylation activity. Emerging evidence indicates that the chromatin-binding and enzymatic modules of UHRF1 do not act in isolation but interplay in a coordinated and regulated manner. Here, we compared two splicing variants (V1, V2) of murine UHRF1 (mUHRF1) with human UHRF1 (hUHRF1). We show that insertion of nine amino acids in a linker region connecting the different TTD and PHD histone modification-binding domains causes distinct H3K9me3-binding behaviour of mUHRF1 V1. Structural analysis suggests that in mUHRF1 V1, in contrast to V2 and hUHRF1, the linker is anchored in a surface groove of the TTD domain, resulting in creation of a coupled TTD-PHD module. This establishes multivalent, synergistic H3-tail binding causing distinct cellular localization and enhanced H3K9me3-nucleosome ubiquitylation activity. In contrast to hUHRF1, H3K9me3-binding of the murine proteins is not allosterically regulated by phosphatidylinositol 5-phosphate that interacts with a separate less-conserved polybasic linker region of the protein. Our results highlight the importance of flexible linkers in regulating multidomain chromatin binding proteins and point to divergent evolution of their regulation.