Backbone 1H, 15N, and 13C resonance assignments and secondary structure of the tollip CUE domain

The Toll-interacting protein (Tollip) is a negative regulator of the Toll-like receptor (TLR)-mediated inflammation response. Tollip is a modular protein that contains an Nterminal Tom1-binding domain (TBD), a central conserved domain 2 (C2), and a C-terminal coupling of ubiquitin to endoplasmic reticulum degradation (CUE) domain. Here, we report the sequence-specific backbone ¹H, ¹⁵N, and ¹³C assignments of the human Tollip CUE domain. The CUE domain was found to be a stable dimer as determined by size-exclusion chromatography and molecular crosslinking studies. Analysis of the backbone chemical shift data indicated that the CUE domain exhibits three helical elements corresponding to 52% of the protein backbone. Circular dichroism spectrum analysis confirmed the helical nature of this domain. Comparison of the location of these helical regions with those reported for yeast CUE domains suggest differences in length for all helical elements. We expect the structural analysis presented here will be the foundation for future studies on the biological significance of the Tollip CUE domain, its molecular interactions, and the mechanisms that modulate its function during the inflammatory response.     

Backbone and side chain 1H, 13C and 15N resonance assignments of the catalytic domain of diphtheria toxin

Diphtheria is a serious upper respiratory tract disease caused by the diphtheria toxin (DT) secreted from the bacteria Corynebacterium diphtheriae. Vaccination is the best way to protect against this infectious disease. Diphtheria vaccines are prepared by isolating, purifying and chemically deactivating DT. Although toxoids have been used for decades in immunization, there is still little understanding at the molecular level of the process of toxoid preparation, and how chemical treatment enhances their immunogenicity. We have undertaken an NMR study of the catalytic domain as a first step in understanding the molecular details involved in vaccine antigen preparation. Here we report a near complete assignment for the backbone and side chain resonances of the diphtheria toxin catalytic domain.     

Backbone and sidechain 1H, 13C and 15N resonance assignments of the RGS domain from human RGS14

We have assigned ¹H, ¹⁵N and ¹³C resonances of the RGS domain from the human RGS14 protein, a multi-domain member of the RGS (Regulators of G-protein signalling) family of proteins, important in the down-regulation of specific G-protein signalling pathways.     

Backbone 1H, 13C, and 15N NMR assignments of the tail domain of vinculin

Vinculin is an essential protein involved in linking the actin cytoskeleton to sites of cell-cell and cell-matrix adhesion. Here we report the majority of the backbone ¹H_N, ¹⁵N, ¹³C_α, ¹³CO, and side chain ¹³C_β NMR resonance assignments of the actin binding tail domain of vinculin (Vt).     

Backbone and side-chain 1H, 13C, 15N resonance assignments of rat lipocalin2

Lipocalin2 plays an important role in the innate immune system. In this article we report the backbone and side-chain resonance assignments of rat lipocalin2 (rLcn2). These assignments provide a basis for determining the structure and dynamics of rLcn2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Backbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda

Lysozyme from lambda bacteriophage (λ lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, λ lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of λ lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes λ lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of λ lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the ¹H, ¹³C and ¹⁵N backbone resonance assignments for λ lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR.     

Backbone and sidechain 1H, 15N and 13C assignments of the NLRP7 pyrin domain

The resonance assignments of the human NLRP7 PYD domain have been determined based on triple-resonance experiments using uniformly [¹³C,¹⁵N]-labeled protein. This assignment is the first step towards the 3D structure determination of the NLRP7 PYD domain.     

Backbone and side-chain 1H, 15N, and 13C resonance assignments of Norwalk virus protease

Norovirus protease cleaves the virus-encoded polyprotein into six mature nonstructural proteins, presenting itself as an essential enzyme for the viral replication as well as an attractive target for the antiviral drug development. A deeper understanding of the structural mechanism of the protease-substrates/inhibitors interactions by means of solution NMR methods would facilitate a rational design of the virus protease inhibitor. We here report the backbone and side-chain resonance assignment of the protease from Norwalk virus, which is the prototype strain of norovirus. The assignment data has been deposited in the BMRB database under the accession number 17523.     

Sequence-specific 1H, 13C, and 15N resonance assignments of GRP1 PH domain

The carboxy-terminal pleckstrin homology (PH) domain recruits GRP1 to the plasma membrane through the specific binding to phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃]. Here, we describe backbone and side chain assignments of the GRP1 PH domain determined by triple resonance experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Backbone and side chain 1H, 15N and 13C assignments of the KSR1 CA1 domain

The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [¹³C, ¹⁵N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain.     

1H, 15N and 13C resonance assignments and monomeric structure of the amino-terminal extracellular domain of epithelial cadherin

Summary E-cadherin is a transmembrane protein that provides Ca²⁺-dependent cell adhesion to epithelial cells. The large majority of the ¹H, ¹⁵N, ¹³C and ¹³CO resonances of a 146-amino acid polypeptide from epithelial (E-) cadherin have been assigned using multidimensional NMR spectroscopy. The structure of the amino-terminal 100 amino acids, corresponding to the first extracellular repeat of E-cadherin [Overduin et al. (1995) Science, 267, 386–389], has been refined. The monomeric state of this isolated domain is demonstrated by light scattering and sedimentation analysis. Seven -strands and two short helices were identified by patterns of NOE cross-peaks, vicinal coupling constants and chemical shift indices. A novel structural motif termed a quasi--helix found in the crystal structure of a neural (N-) cadherin domain [Shapiro et al. (1995) Nature, 374, 327–337] is characterized in detail for the first time by NMR. Slowly exchanging amides were concentrated in the -sheet region and quasi--helix. The -barrel fold of the cadherin domain is topologically similar to the immunoglobulin fold. Comparison of this solution structure to the crystallized dimers of the N-terminal pair of E-cadherin domains [Nagar et al. (1996) Nature, 380, 360–364] and of the homologous single domain of N-cadherin reveals a conserved cadherin fold with minor structural differences, which can be accounted for by differences in metal ligation and oligomeric state.Abbreviations cad extracellular cadherin repeat - CAM cell adhesion molecule - CSI chemical shift index - DTT dithiothreitol - E-cadherin epithelial cadherin - N-cadherin neural cadherin - NOE nuclear Overhauser enhancement - PFG pulsed field gradient - rmsd root-mean-square deviation     

Backbone 1H, 15N, and 13C resonance assignments of the Helicobacter pylori acyl carrier protein

One of the small proteins from Helicobacter pylori, acyl carrier protein (ACP), was investigated by NMR. ACP is related to various cellular processes, especially with the biosynthesis of fatty acid. The basic NMR resonance assignment is a prerequisite for the validation of a heterologous protein interaction with ACP in H. pylori. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the H. pylori ACP are reported using double- and triple-resonance techniques. About 97% of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 76 of the 78 non-proline residues are clarified through sequential- and specific- assignments. In addition, four helical regions were clearly identified on the basis of the resonance assignments.     

1H, 13C and 15N resonance assignments of domain 1 of receptor associated protein

The 39 kDa receptor associated protein (RAP) is a modular protein consisting of multiple domains. There has been no x-ray crystal structure of RAP available and the full-length protein does not behave well in a NMR tube. To elucidate the 3D structure of the RAP, we undertook structure determination of individual domains of the RAP. As the first step, here we report the nearly complete assignments of the ¹H, ¹³C and ¹⁵N chemical shift signals of domain 1 of the RAP.     

1H, 13C and 15N resonance assignments of human H-REV107 N-terminal domain

Human H-REV107 protein is the representative of a novel class II tumor suppressor family, which is lost in tumor cells and can induce cell death after restoration. The NMR assignments of the H-REV107 N-terminal domain are essential for its solution structure determination.     

1H, 13C and 15N resonance assignments of SARS-CoV main protease N-terminal domain

The main protease (M^pro) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. SARS-CoV M^pro is composed of a catalytic N-terminal domain and an α-helical C-terminal domain linked by a long loop. Even though the N-terminal domain of SARS-CoV M^pro adopts a similar chymotrypsin-like fold as that of piconavirus 3C protease, the extra C-terminal domain is required for SARS-CoV M^pro to be enzymatically active. Here, we reported the NMR assignments of the SARS-CoV M^pro N-terminal domain alone, which are essential for its solution structure determination.     

Backbone and side-chain resonance assignments (1H, 15N and 13C) of the ubiquitin homology domain of mouse BAG-1

BAG-1, an important regulatory protein associates with several signaling molecules and is capable of suppressing apoptosis. A 97-amino acid segment that includes the ubiquitin homology domain of mouse BAG-1 interacts with the cytoplasmic tail domain of proHB-EGF, and this interaction is likely to have functional significance. Here we report the backbone and side-chain resonance assignments for this 97-amino acid segment of mouse BAG-1. The assignment data has been deposited in the BMRB database under the accession number 18416.     

1H, 13C and 15N resonance assignments of the pyrin domain from human PYNOD

PYNOD is a novel protein belonging to a large family of proteins containing the nucleotide-binding and oligomerization domain (NOD) involved in inflammation and apoptosis. Human PYNOD inhibits inflammatory response mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). Here we report the ¹H, ¹³C and ¹⁵N resonance assignments and secondary structure identification of the pyrin domain (PYD) of human PYNOD as the first step towards elucidating the structural basis of the anti-inflammatory activity of PYNOD.