SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) are transcriptional corepressors that play an essential role in the regulation of development and metabolism. This role is achieved, in part, through the recruitment of a key histone deacetylase (HDAC3), which is itself indispensable for cell viability. The assembly of HDAC3 with the deacetylase activation domain (DAD) of SMRT and NCoR is required for activation of the otherwise inert deacetylase. The DAD comprises an N-terminal DAD-specific motif and a C-terminal SANT (SWI3/ADA2/NCoR/TFIIIB)-like domain. We report here the solution structure of the DAD from SMRT, which reveals a four-helical structure. The DAD differs from the SANT (and MYB) domains in that (i) it has an additional N-terminal helix and (ii) there is a notable hydrophobic groove on the surface of the domain. Structure-guided mutagenesis, combined with interaction assays, showed that residues in the vicinity of the hydrophobic groove are required for interaction with (and hence activation of) HDAC3. Importantly, one surface-exposed lysine is required for activation of HDAC3, but not for interaction. This lysine may play a uniquely important role in the mechanism of activating HDAC3.

The nuclear receptor Nurr1 is a transcription factor essential for the development of midbrain dopaminergic neurons in vertebrates. Recent crystal structures of the Nurr1 ligand binding domain (LBD) and the Drosophila orthologue dHR38 revealed that, although these receptors share the classical LBD architecture, they lack a ligand binding cavity. This volume is instead filled with bulky hydrophobic side chains. Furthermore the “canonical” non-polar co-regulator binding groove is filled with polar side chains; thus, the regulation of transcription by this sub-family of nuclear receptor LBDs may be mediated by some other interaction surface on the LBD. We report here the identification of a novel co-regulator interface on the LBD of Nurr1. We used an NMR footprinting strategy that facilitates the identification of an interaction surface without the need of a full assignment. We found that non-polar peptides derived from the co-repressors SMRT and NCoR bind to a hydrophobic patch on the LBD of Nurr1. This binding surface involves a groove between helices 11 and 12. Mutations in this site abolish activation by the Nurr1 LBD. These findings give insight into the unique mechanism of action of this class of nuclear receptors.

The total synthesis and structural characterization of the MHCII-associated p41 invariant chain fragment (P41icf) is described. P41icf plays a crucial role in the maturation of MHC class II molecules and antigen processing, acting as a highly selective cathepsin L inhibitor. P41icf synthesis was achieved using a combined solid-phase/solution approach. The entire molecule (65 residues, 7246 Da unprotected) was assembled in solution from fully protected peptides in the size range of 10 residues. After deprotection, oxidative folding in carefully adjusted experimental conditions led to the completely folded and functional P41icf with a disulfide pairing identical to that of native P41icf. CD, NMR, and surface plasmon resonance (SPR) were used for the structural and functional characterization of synthetic P41icf. CD thermal denaturation showed clear cooperative behavior. Tight cathepsin L binding was demonstrated by SPR. (1)H NMR spectroscopy at 800 MHz of unlabeled P41icf was used to solve the three-dimensional structure of the molecule. P41icf behaves as a well-folded protein domain with a topology very close to the crystallographic cathepsin L-bound form.

An alpha-L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal alpha-L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first alpha-L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an alpha-L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without alpha-L-fucosidase activity. Pea plants had endogenous alpha-L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated alpha-L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the alpha-L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.

A simplified strategy is described for the assignment of disulfide bonds in proteins of medium to high molecular mass (10-30 kDa). The method combines the use of high-performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) and HPLC with UV diode-array detection (HPLC diode array). The denatured protein is subjected to proteolysis and the peptide mixture is divided into three fractions: (i) underivatized peptides, (ii) ethylpyridylated peptides, and (iii) reduced and ethylpyridylated peptides. The three peptide ensembles are then subjected to chromatographic and spectroscopic analysis. A systematic methodology is described to analyze the large amount of data obtained. The method was applied to the localization of disulfide bonds in alpha -L-fucosidase from pea. The two disulfide bonds were located between residues Cys(64) and Cys(109) and between Cys(162) and Cys(169), while Cys(127) was free.

Fuc‐9 is the mature form of a vacuolar α‐L;‐fucosidase enzyme which seems to play an important role in plant growth regulation. Fuc‐9 is a 202‐residue protein containing five Cys residues located at positions 64, 109, 127, 162 and 169. In this study, the disulfide structure of Fuc‐9 was determined by MALDI‐TOF mass spectrometry (MS), with minimal clean‐up of the samples and at a nanomolar scale. Two strategies, based on a specific chemical cleavage (with 2‐nitro‐5‐thiocyanobenzoic acid and alkaline conditions) at the Cys residues and modification of Cys residues by acrylamide/deuterium labeled acrylamide alkylation, were used. Using these methods, the disulfide pairings Cys64‐Cys109 and Cys162‐Cys169 could be established. The advantages and limitations of our experimental approach are discussed. Copyright © 2001 European Peptide Society and John Wiley & Sons, Ltd.