Biomedical subjects

Joanne M O'Leary

Publications and source records attributed to Joanne M O'Leary.

6 recordsLinked to original sources

PubMed2004

Solution structure and dynamics of a prototypical chordin-like cysteine-rich repeat (von Willebrand Factor type C module) from collagen IIA.

Chordin-like cysteine-rich (CR) repeats (also referred to as von Willebrand factor type C (VWC) modules) have been identified in approximately 200 extracellular matrix proteins. These repeats, named on the basis of amino acid conservation of 10 cysteine residues, have been shown to bind members of the transforming growth factor-beta (TGF-beta) superfamily and are proposed to regulate growth factor signaling. Here we describe the intramolecular disulfide bonding, solution structure, and dynamics of a prototypical chordin-like CR repeat from procollagen IIA (CR(ColIIA)), which has been previously shown to bind TGF-beta1 and bone morphogenetic protein-2. The CR(ColIIA) structure manifests a two sub-domain architecture tethered by a flexible linkage. Initial structures were calculated using RosettaNMR, a de novo prediction method, and final structure calculations were performed using CANDID within CYANA. The N-terminal region contains mainly beta-sheet and the C-terminal region is more irregular with the fold constrained by disulfide bonds. Mobility between the N- and C-terminal sub-domains on a fast timescale was confirmed using NMR relaxation measurements. We speculate that the mobility between the two sub-domains may decrease upon ligand binding. Structure and sequence comparisons have revealed an evolutionary relationship between the N-terminal sub-domain of the CR module and the fibronectin type 1 domain, suggesting that these domains share a common ancestry. Based on the previously reported mapping of fibronectin binding sites for vascular endothelial growth factor to regions containing fibronectin type 1 domains, we discuss the possibility that this structural homology might also have functional relevance.

Algorithms↗

PubMed2004

N-Terminal domain linkage modulates the folding properties of protein S epidermal growth factor-like modules.

Protein S interacts with activated protein C to play a crucial role in blood anticoagulation, and protein S deficiency is associated with increased risk of thrombosis. Despite the large volume of functional data available for this protein, no atomic resolution structure data have yet been reported. This is due at least in part to difficulties encountered when trying to produce fragments dissected from the intact protein; however, a few successful strategies have been described. In this research we have expressed a number of constructs containing protein S epidermal growth factor-like (EGF) domains 1 and 2 in Escherichia coli and Pichia pastoris. None of the proteins produced was stably folded as assayed by solution nuclear magnetic resonance spectroscopy. We therefore constructed a series of non-native protein S EGF concatemers to investigate the role of pairwise domain linkage in domain folding. Our results demonstrate that N-terminal domain linkage can either positively or negatively impact on the refolding of an adjacent domain. Furthermore, analysis of the NMR data for EGF3-4 reveals the expected interdomain NOEs that are characteristic of an extended arrangement of calcium-binding EGF domains and a similar average [(1)H]-(15)N heteronuclear NOE value for each of the two domains. These results provide the first data in support of protein S EGF3-4 adopting the same extended domain orientation as observed for the functionally distinct proteins fibrillin-1 and the low-density lipoprotein receptor. The results also have important implications for future studies, particularly when a dissection approach is used, of tandem EGF domains from protein S and other proteins.

Amino Acid Sequence↗

PubMed2004

Identification and removal of O-linked and non-covalently linked sugars from recombinant protein produced using Pichia pastoris.

The use of the methylotrophic yeast Pichia pastoris for large-scale recombinant production of proteins for therapeutic uses and/or biophysical characterisation has been gaining popularity. Here we describe the use of this organism for the production of a von Willebrand factor C domain from procollagen IIA for solution NMR studies. In this research, we specifically identified sites of O-linked glycosylation on the expressed protein, although the native protein is not glycosylated. We demonstrated that it was possible to remove the oligosaccharides by enzymatic digestion, however this approach proved to be prohibitively expensive for the scale of production required for high-resolution structural studies by NMR spectroscopy. After removal of the O-linked glycosylation sites by site-directed mutagenesis, we confirmed that the protein was no longer covalently glycosylated. However, analysis by 1H- and 13C-edited spectroscopy identified the presence of non-covalently associated glycans which were removed by lectin affinity chromatography. We have synthesised methods for the identification and removal of both covalently and non-covalently bound oligosaccharides from heterologous protein expressed in P. pastoris.

Chromatography, Affinity↗

PubMed2004

Backbone dynamics of complement control protein (CCP) modules reveals mobility in binding surfaces.

The regulators of complement activation (RCA) are critical to health and disease because their role is to ensure that a complement-mediated immune response to infection is proportionate and targeted. Each protein contains an uninterrupted array of from four to 30 examples of the very widely occurring complement control protein (CCP, or sushi) module. The CCP modules mediate specific protein-protein and protein-carbohydrate interactions that are key to the biological function of the RCA and, paradoxically, provide binding sites for numerous pathogens. Although structural and mutagenesis studies of CCP modules have addressed some aspects of molecular recognition, there have been no studies of the role of molecular dynamics in the interaction of CCP modules with their binding partners. NMR has now been used in the first full characterization of the backbone dynamics of CCP modules. The dynamics of two individual modules-the 16th of the 30 modules of complement receptor type 1 (CD35), and the N-terminal module of membrane cofactor protein (CD46)-as well as their solution structures, are compared. Although both examples share broadly similar three-dimensional structures, many structurally equivalent residues exhibit different amplitudes and timescales of local backbone motion. In each case, however, regions of the module-surface implicated by mutagenesis as sites of interactions with other proteins include several mobile residues. This observation suggests further experiments to explore binding mechanisms and identify new binding sites.

Amino Acid Sequence↗

PubMed2004

Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris.

The methylotrophic yeast Pichia pastoris is now an established expression system for the production of recombinant protein for nuclear magnetic resonance (NMR) studies. It is capable of expressing high levels of heterologous proteins and possesses the ability to perform many of the posttranslational modifications of higher eukaryotes. Here, we describe efficient methods for the production of uniformly 13C,15N-labeled proteins in shake flasks and of uniformly 13C,15N-labeled and 2H,13C,15N-labeled proteins in fermenters. We also provide details of two chromatographic procedures, cation exchange and concanavalin A lectin affinity, that have proved useful in purifying P. pastoris-expressed proteins for NMR studies.

Carbon Isotopes↗

PubMed2003

Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta.

Almost all TGF-beta is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-beta binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-beta cytokine. LAP is the TGF-beta pro-peptide that is cleaved intracellularly prior to secretion, and TGF-beta binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-beta. Previous studies have revealed that the LTBP-LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3(LTBP1). Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs.

Amino Acid Sequence↗