No abstract

The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot be replaced by other members of the same family to induce pluripotency. To understand the unique role of Oct4 in reprogramming, we determined the structure of its POU domain bound to DNA. We show that the linker between the two DNA-binding domains is structured as an α-helix and exposed to the protein's surface, in contrast to the unstructured linker of Oct1. Point mutations in this α-helix alter or abolish the reprogramming activity of Oct4, but do not affect its other fundamental properties. On the basis of mass spectrometry studies of the interactome of wild-type and mutant Oct4, we propose that the linker functions as a protein-protein interaction interface and plays a crucial role during reprogramming by recruiting key epigenetic players to Oct4 target genes. Thus, we provide molecular insights to explain how Oct4 contributes to the reprogramming process.

As in many other animals, the primordial germ cells (PGCs) in avian and reptile embryos are specified in positions distinct from the positions where they differentiate into sperm and egg. Unlike in other organism however, in these embryos, the PGCs use the vascular system as a vehicle to transport them to the region of the gonad where they exit the blood vessels and reach their target. To determine the molecular mechanisms governing PGC migration in these species, we have investigated the role of the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) in guiding the cells towards their target in the chick embryo. We show that sdf-1 mRNA is expressed in locations where PGCs are found and towards which they migrate at the time they leave the blood vessels. Ectopically expressed chicken SDF-1alpha led to accumulation of PGCs at those positions. This analysis, as well as analysis of gene expression and PGC behavior in the mouse embryo, suggest that in both organisms, SDF-1 functions during the second phase of PGC migration, and not at earlier phases. These findings suggest that SDF-1 is required for the PGCs to execute the final migration steps as they transmigrate through the blood vessel endothelium of the chick or the gut epithelium of the mouse.