Structure of Prototypic Peptide Transporter DtpA from E. coli in Complex with Valganciclovir Provides Insights into Drug Binding of Human PepT1.

Members of the solute carrier 15 family (SLC15) transport di- and tripeptides as well as peptidomimetic drugs across the cell membrane. Structures of bacterial homologues have provided valuable information on the binding and transport of their natural substrates, but many do not transport medically relevant drugs. In contrast, a homologue from Escherichia coli, DtpA (dipeptide and tripeptide permease), shows a high similarity to human PepT1 (SLC15A1) in terms of ligand selectivity and transports a similar set of drugs. Here, we present the crystal structure of DtpA in ligand-free form (at 3.30 Å resolution) and in complex with the antiviral prodrug valganciclovir (at 2.65 Å resolution) supported by biochemical data. We show that valganciclovir unexpectedly binds with the ganciclovir moiety mimicking the N-terminal residue of a canonical peptide substrate. On the basis of a homology model we argue that this binding mode also applies to the human PepT1 transporter. Our results provide new insights into the binding mode of prodrugs and will assist the rational design of drugs with improved absorption rates.

Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research.

Saposin-derived lipid nanoparticles (SapNPs) are a new alternative tool for membrane protein reconstitution. Here we demonstrate the potential and advantages of SapNPs. We show that SapA has the lowest lipid specificity for SapNP formation. These nanoparticles are modular and offer a tunable range of size and composition depending on the stoichiometric ratio of lipid and saposin components. They are stable and exhibit features typical of lipid-bilayer systems. Our data suggest that SapNPs are versatile and can adapt to membrane proteins of various sizes and architectures. Using SapA and various types of lipids we could reconstitute membrane proteins of different transmembrane cross-sectional areas (from 14 to 56 transmembrane α helices). SapNP-reconstituted proteins bound their respective ligands and were more heat stable compared with the detergent-solubilized form. Moreover, SapNPs encircle membrane proteins in a compact way, allowing structural investigations of small membrane proteins in a detergent-free environment using small-angle X-ray scattering.

Molecular insights into substrate recognition and catalytic mechanism of the chaperone and FKBP peptidyl-prolyl isomerase SlyD.

BACKGROUND: Peptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide. RESULTS: We have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through ß-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline ß-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk. CONCLUSIONS: We found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site.

Rapid divergence and high diversity of miRNAs and miRNA targets in the Camelineae.

MicroRNAs (miRNAs) are short RNAs involved in gene regulation through translational inhibition and transcript cleavage. After processing from imperfect fold-back structures, miRNAs are incorporated into RNA-induced silencing complexes (RISCs) before targeting transcripts with varying degrees of complementarity. Some miRNAs are evolutionarily deep-rooted, and sequence complementarity with their targets is maintained through purifying selection. Both Arabidopsis and Capsella belong to the tribe Camelineae in the Brassicaceae, with Capsella rubella serving as an outgroup to the genus Arabidopsis. The genome sequence of C. rubella has recently been released, which allows characterization of its miRNA complement in comparison with Arabidopsis thaliana and Arabidopsis lyrata. Through next-generation sequencing, we identify high-confidence miRNA candidates specific to the C. rubella lineage. Only a few lineage-specific miRNAs have been studied for evolutionary constraints, and there have been no systematic studies of miRNA target diversity within or divergence between closely related plant species. Therefore we contrast sequence variation in miRNAs and their targets within A. thaliana, and between A. thaliana, A. lyrata and C. rubella. We document a surprising amount of small-scale variation in miRNA-target pairs, where many miRNAs are predicted to have species-specific targets in addition to ones that are shared between species. Our results emphasize that the transitive nature of many miRNA-target pairs can be observed even on a relatively short evolutionary time-scale, with non-random occurrences of differences in miRNAs and their complements in the miRNA precursors, the miRNA* sequences.