NMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains.

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L

Random coil conformation of a Gly/Ala-rich insert in IκBα excludes structural stabilization as the mechanism for protection against proteasomal degradation

AbstractPeptide segments of multiple glycine and alanine residues prevent the proteolytic degradation of ubiquitinated proteins by the proteasome. The structure of a Gly/Ala-rich insert in IκBα was probed by nuclear magnetic resonance (NMR) spectroscopy, comparing IκBα samples with and without Gly/Ala-rich insert. Narrow 1H-NMR resonances at chemical shifts indicative of random coil conformations were observed in the difference spectrum. circular dichroism (CD) measurements further confirm that the mechanism of protection against proteolytic degradation is not based on structural transition or stabilization caused by the Gly/Ala-rich segment. In addition, most of the N- and C-terminal residues outside the ankyrin repeats in wild-type IκBα were found to be flexibly disordered

Intramolecular C-H⋯O hydrogen bonding in 1,4-dihydropyridine derivatives

The diastereotopy of the methylene protons at positions 2 and 6 in 1,4-dihydropiridine derivatives with various substituents has been investigated. NMR spectroscopy and quantum chemistry calculations show that the CH⋯O intramolecular hydrogen bond is one of the factors amplifying the chemical shift differences in the 1H-NMR spectra.publishersversionPeer reviewe

A novel zinc-binding fold in the helicase interaction domain of the Bacillus subtilis DnaI helicase loader

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of DnaI to the hexameric helicase from B. stearothermophilus, which is a close homologue of the corresponding much less stable B. subtilis helicase

NMR structure of the WIF domain of the human Wnt-inhibitory factor-1

The human Wnt-binding protein Wnt-inhibitory factor-1 (WIF-1) comprises an N-terminal WIF module followed by five EGF-like repeats. Here we report the three-dimensional structure of the WIF domain of WIF-1 determined by NMR spectroscopy. The fold consists of an eight-stranded β-sandwich reminiscent of the immunoglobulin fold. Residual detergent (Brij-35) used in the refolding protocol was found to bind tightly to the WIF domain. The binding site was identified by intermolecular nuclear Overhauser effects observed between the WIF domain and the alkyl chain of the detergent. The results point to a possible role of WIF domains as a recognition motif of Wnt and Drosophila Hedgehog proteins that are activated by palmitoylation

Data from: Intramolecular hydrogen bonds in 1,4-dihydropyridine derivatives

1,4- dihydropyridine (1,4-DHP) derivatives have been synthesized and characterized by 1H, 13C, 15N NMR spectroscopy, secondary H/D 13C isotope shifts, variable temperature 1H NMR experiments and quantum-chemical calculation. The intramolecular hydrogen-bonds NH•••O=C and CH•••O=C in these compounds were established by NMR and quantum-chemical studies. The downfield shift of the NH proton, accompanied by the upfield shift of the 15N NMR signals, the shift to the higher wave numbers of the NH stretching vibration in the IR spectra and the increase of the 1J(15N,1H) values may indicate the shortening of the N-H bond length upon intramolecular NH•••O=C hydrogen bond formation

NMR Structure of Citrobacter freundii AmpD, Comparison with Bacteriophage T7 Lysozyme and Homology with PGRP Domains

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of β-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resis

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions

Solution Structure of the R3H Domain from Human Sμbp-2

The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA. In this work the 3D structure of the R3H domain from human Sμbp-2 was de