SUMMARY Mammalian cells express dozens of iron-containing proteins, yet little is known about the mechanism of metal ligand incorporation. Human poly (rC) binding protein 1 (PCBP1) is an iron chaperone that binds iron and delivers it to ferritin, a cytosolic iron storage protein. We have identified the iron-dependent prolyl hydroxylases (PHDs) and asparaginyl hydroxylase (FIH1) that modify hypoxia-inducible factor α (HIFα) as targets of PCBP1. Depletion of PCBP1 or PCBP2 in cells led to loss of PHD activity, manifested by reduced prolyl hydroxylation of HIF1α, impaired degradation of HIF1α through the VHL/proteasome pathway, and accumulation of active HIF1 transcription factor. PHD activity was restored in vitro by addition of excess Fe(II), or purified Fe-PCBP1, and PCBP1 bound to PHD2 and FIH1 in vivo. These data indicated that PCBP1 was required for iron incorporation into PHD and suggest a broad role for PCBP1 and 2 in delivering iron to cytosolic nonheme iron enzymes.

Transient receptor potential canonical (TRPC) proteins form nonselective cation channels that play physiological roles in a wide variety of cells. Despite growing evidence supporting the therapeutic potential of TRPC6 inhibition in treating pathological cardiac and renal conditions, mechanistic understanding of TRPC6 function and modulation remains obscure. Here we report cryo-EM structures of TRPC6 in both antagonist-bound and agonist-bound states. The structures reveal two novel recognition sites for the small-molecule modulators corroborated by mutagenesis data. The antagonist binds to a cytoplasm-facing pocket formed by S1-S4 and the TRP helix, whereas the agonist wedges at the subunit interface between S6 and the pore helix. Conformational changes upon ligand binding illuminate a mechanistic rationale for understanding TRPC6 modulation. Furthermore, structural and mutagenesis analyses suggest several disease-related mutations enhance channel activity by disrupting interfacial interactions. Our results provide principles of drug action that may facilitate future design of small molecules to ameliorate TRPC6-mediated diseases.

Background:The functional role of gene duplicates in a bacterial metabolite transport system is unknown. Results: Duplicated genes have similar ligand specificities, but affinities differ by three orders of magnitude. Conclusion: This expands the ability to respond to nutrient stress and maximizes transport of preferentially metabolized substrates. Significance: Functionalization of gene duplicates in transport pathways allows bacteria to adapt to varying nutrient flux.

BackgroundMembers of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to specifically bind a chemically diverse range of ligands. This paradigm of PBP ligand binding specificity was recently altered when the structure of the Thermotoga maritima cellobiose-binding protein (tmCBP) was solved. The tmCBP binding site is bipartite, comprising a canonical solvent-excluded region (subsite one), adjacent to a solvent-filled cavity (subsite two) where specific and semi-specific ligand recognition occur, respectively.ResultsA molecular level understanding of binding pocket adaptation mechanisms that simultaneously allow both ligand specificity at subsite one and promiscuity at subsite two has potentially important implications in ligand binding and drug design studies. We sought to investigate the determinants of ligand binding selectivity in tmCBP through biophysical characterization of tmCBP in the presence of varying β-glucan oligosaccharides. Crystal structures show that whilst the amino acids that comprise both the tmCBP subsite one and subsite two binding sites remain fixed in conformation regardless of which ligands are present, the rich hydrogen bonding potential of water molecules may facilitate the ordering and the plasticity of this unique PBP binding site.ConclusionsThe identification of the roles these water molecules play in ligand recognition suggests potential mechanisms that can be utilized to adapt a single ligand binding site to recognize multiple distinct ligands.

Protein controlled iron homeostasis is essential for maintaining appropriate levels and availability of metal within cells. Recently, two iron chaperones have been discovered that direct metal within two unique pathways: (1) mitochondrial iron–sulfur (Fe–S) cluster assembly and (2) within the ferritin iron storage system. Although structural and functional details describing how these iron chaperones operate are emerging, both share similar iron binding affinities and metal–ligand site structures that enable them to bind and release Fe2+ to specific protein partners. Molecular details related to iron binding and delivery by these chaperones will be explored within this review.