Click chemistry-based tracking reveals putative cell wall-located auxin binding sites in expanding cells

Abstract Auxin is a key plant regulatory molecule, which acts upon a plethora of cellular processes, including those related to cell differentiation and elongation. Despite the stunning progress in all disciplines of auxin research, the mechanisms of auxin-mediated rapid promotion of cell expansion and underlying rearrangement of cell wall components are poorly understood. This is partly due to the limitations of current methodologies for probing auxin. Here we describe a click chemistry-based approach, using an azido derivative of indole-3-propionic acid. This compound is as an active auxin analogue, which can be tagged in situ. Using this new tool, we demonstrate the existence of putative auxin binding sites in the cell walls of expanding/elongating cells. These binding sites are of protein nature but are distinct from those provided by the extensively studied AUXIN BINDING PROTEIN 1 (ABP1). Using immunohistochemistry, we have shown the apoplastic presence of endogenous auxin epitopes recognised by an anti-IAA antibody. Our results are intriguingly in line with previous observations suggesting some transcription-independent (non-genomic) activity of auxin in cell elongation

A cyclic peptidic serine protease inhibitor:increasing affinity by increasing peptide flexibility

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden

Site-selective modification of proteins

The present invention concerns a method for site-selective modification of a target protein or peptide by use of an acylation tag comprising a single lysine residue and at least three histidine residues. Upon contact with an acylating reagent, the target protein or peptide becomes modified at the ε-amine of the lysine residue of the acylation tag.</p