Distinct conformational and energetic features define the specific recognition of (di)aromatic peptide motifs by PEX14.

The cycling import receptor PEX5 and its membrane-located binding partner PEX14 are key constituents of the peroxisomal import machinery. Upon recognition of newly synthesized cargo proteins carrying a peroxisomal targeting signal type 1 (PTS1) in the cytosol, the PEX5/cargo complex docks at the peroxisomal membrane by binding to PEX14. The PEX14 N-terminal domain (NTD) recognizes (di)aromatic peptides, mostly corresponding to Wxxx(F/Y)-motifs, with nano-to micromolar affinity.

The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold.

Squash is an RNA aptamer that strongly activates the fluorescence of small-molecule analogs of the fluorophore of green fluorescent protein (GFP). Unlike other fluorogenic aptamers, isolated de novo from random-sequence RNA, Squash was evolved from the bacterial adenine riboswitch to leverage its optimized in vivo folding and stability. We now report the 2.

Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility.

Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range.

NMR Analysis of Apo Glutamine-Binding Protein Exposes Challenges in the Study of Interdomain Dynamics.

Glutamine-binding protein (GlnBP) displays an apo, "open" and a holo, "closed" crystal form, mutually related by a rigid-body reorientation of its domains. A fundamental question about such large-scale conformational transitions, whether the closed state exists in the absence of ligand, is controversial in the case of GlnBP. NMR observations have indicated no evidence of the closed form, whereas experimentally validated computations have suggested a remarkable ca.

Conformational Ensemble of Disordered Proteins Probed by Solvent Paramagnetic Relaxation Enhancement (sPRE).

Characterization of the conformational ensemble of disordered proteins is highly important for understanding protein folding and aggregation mechanisms, but remains a computational and experimental challenge owing to the dynamic nature of these proteins. New observables that can provide unique insights into transient residual structures in disordered proteins are needed. Here using denatured ubiquitin as a model system, NMR solvent paramagnetic relaxation enhancement (sPRE) measurements provide an accurate and highly sensitive probe for detecting low populations of residual structure in a disordered protein.

Structural basis for terminal loop recognition and stimulation of pri-miRNA-18a processing by hnRNP A1.

Post-transcriptional mechanisms play a predominant role in the control of microRNA (miRNA) production. Recognition of the terminal loop of precursor miRNAs by RNA-binding proteins (RBPs) influences their processing; however, the mechanistic basis for how levels of individual or subsets of miRNAs are regulated is mostly unexplored. We previously showed that hnRNP A1, an RBP implicated in many aspects of RNA processing, acts as an auxiliary factor that promotes the Microprocessor-mediated processing of pri-mir-18a.

Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins.

Sirt1 is an NAD-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1.

A novel Pex14 protein-interacting site of human Pex5 is critical for matrix protein import into peroxisomes.

Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14.

Efficient detection of hydrogen bonds in dynamic regions of RNA by sensitivity-optimized NMR pulse sequences.

Improved Sensitivity: Efficient NMR experiments are presented for determining the secondary structure in large and dynamic RNAs using J-couplings across hydrogen bonds. The experiments provide up to eight-fold improved sensitivity and thus enable detection of base pairs in dynamic regions even in large RNAs.

hnRNP A1 proofreads 3' splice site recognition by U2AF.

One of the earliest steps in metazoan pre-mRNA splicing involves binding of U2 snRNP auxiliary factor (U2AF) 65 KDa subunit to the polypyrimidine (Py) tract and of the 35 KDa subunit to the invariant AG dinucleotide at the intron 3' end. Here we use in vitro and in vivo depletion, as well as reconstitution assays using purified components, to identify hnRNP A1 as an RNA binding protein that allows U2AF to discriminate between pyrimidine-rich RNA sequences followed or not by a 3' splice site AG. Biochemical and NMR data indicate that hnRNP A1 forms a ternary complex with the U2AF heterodimer on AG-containing/uridine-rich RNAs, while it displaces U2AF from non-AG-containing/uridine-rich RNAs, an activity that requires the glycine-rich domain of hnRNP A1.

Structural basis for competitive interactions of Pex14 with the import receptors Pex5 and Pex19.

Protein import into peroxisomes depends on a complex and dynamic network of protein-protein interactions. Pex14 is a central component of the peroxisomal import machinery and binds the soluble receptors Pex5 and Pex19, which have important function in the assembly of peroxisome matrix and membrane, respectively. We show that the N-terminal domain of Pex14, Pex14(N), adopts a three-helical fold.