Publications by authors named "Julia A Hubbard"

9 Publications

  • Page 1 of 1

Backbone resonance assignments of the catalytic and regulatory domains of Ca/calmodulin-dependent protein kinase 1D.

Biomol NMR Assign 2020 10 13;14(2):221-225. Epub 2020 Jun 13.

Department of Biochemistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada.

The CaMK subfamily of Ser/Thr kinases are regulated by calmodulin interactions with their C-terminal regions. They are exemplified by Ca/calmodulin dependent protein kinase 1δ which is known as CaMK1D, CaMKIδ or CKLiK. CaMK1D mediates intracellular signalling downstream of Ca influx and thereby exhibits amplifications of Casignals and polymorphisms that have been implicated in breast cancer and diabetes. Here we report the backbone H, C, N assignments of the 38 kDa human CaMK1D protein in its free state, including both the canonical bi-lobed kinase fold as well as the autoinhibitory and calmodulin binding domains.
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http://dx.doi.org/10.1007/s12104-020-09950-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462902PMC
October 2020

Production and Crystallization of Full-Length Human AMP-Activated Protein Kinase (α1β1γ1).

Methods Mol Biol 2018 ;1732:1-14

The Francis Crick Institute, London, UK.

Determination of the crystal structure of AMP-activated protein kinase (AMPK) is fundamental to understanding its biological function and role in a number of diseases related to energy metabolism including type 2 diabetes, obesity, and cancer. We describe methods for the expression and purification of a human full-length active AMPK complex that is suitable for biochemical and structural analyses, followed by methods for its crystallization in complex with small molecule activators. Quality control of the purified protein by functional and biophysical analysis was an essential part of the process enabling the achievement of crystals of the full-length protein capable of being used for high-resolution structure determination by X-ray diffraction. X-ray structures have been determined of both phosphorylated and non-phosphorylated forms of full-length human AMPK α1β1γ1.
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http://dx.doi.org/10.1007/978-1-4939-7598-3_1DOI Listing
January 2019

Unlocking the secrets of the gatekeeper: methods for stabilizing and crystallizing GPCRs.

Biochim Biophys Acta 2013 Nov 18;1828(11):2583-91. Epub 2013 Jul 18.

Department of Life Sciences, Imperial College London, SW7 2AZ, UK.

G-protein coupled receptors (GPCRs) are integral membrane cell surface receptors with key roles in mediating the cellular responses to a wide range of biologically relevant molecules including hormones, neurotransmitters and importantly the majority of currently available drugs. The first high-resolution, X-ray crystallographic structure of a GPCR, that of rhodopsin, was obtained in 2000. It took a further seven years for the next structure, that of the β2 adrenergic receptor. Remarkably, at the time of writing, there have been an astonishing 18 further independent high-resolution GPCR structures published in the last five years (overall total of 68 structures in different conformations or bound to different ligands). Of particular note is the recent structure of the β2 adrenergic receptor in complex with its cognate heterotrimeric G-protein revealing for the first time molecular details of the interaction between a GPCR and the complete G-protein. Together these structures have provided unprecedented detail into the mechanism of action of these incredibly important proteins. This review describes several key methodological advances that have made such extraordinarily fast progress possible.
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http://dx.doi.org/10.1016/j.bbamem.2013.07.013DOI Listing
November 2013

Novel macrocyclic HCV NS3 protease inhibitors derived from α-amino cyclic boronates.

Bioorg Med Chem Lett 2010 Oct 10;20(19):5695-700. Epub 2010 Aug 10.

Anacor Pharmaceuticals, Inc., 1020 E. Meadow Circle, Palo Alto, CA 94303, USA.

A novel series of P2-P4 macrocyclic HCV NS3/4A protease inhibitors with α-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around α-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.
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http://dx.doi.org/10.1016/j.bmcl.2010.08.022DOI Listing
October 2010

Synthesis and evaluation of novel alpha-amino cyclic boronates as inhibitors of HCV NS3 protease.

Bioorg Med Chem Lett 2010 Jun 20;20(12):3550-6. Epub 2010 May 20.

Anacor Pharmaceuticals, Inc., Palo Alto, CA 94303, USA.

We have designed and synthesized a novel series of alpha-amino cyclic boronates and incorporated them successfully in several acyclic templates at the P1 position. These compounds are inhibitors of the HCV NS3 serine protease, and structural studies show that they inhibit the NS3 protease by trapping the Ser-139 hydroxyl group in the active site. Synthetic methodologies and SARs of this series of compounds are described.
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http://dx.doi.org/10.1016/j.bmcl.2010.04.129DOI Listing
June 2010

Selective and dual action orally active inhibitors of thrombin and factor Xa.

Bioorg Med Chem Lett 2007 May 30;17(10):2927-30. Epub 2007 Mar 30.

GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.

The synthetic entry to new classes of dual fXa/thrombin and selective thrombin inhibitors with significant oral bioavailability is described. This was achieved through minor modifications to the sulfonamide group in our potent and selective fXa inhibitor (E)-2-(5-chlorothien-2-yl)-N-{(3S)-1-[(1S)-1-methyl-2-(morpholin-4-yl)-2-oxoethyl]-2-oxopyrrolidin-3-yl}ethenesulfonamide and these observed activity changes have been rationalised using structural studies.
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http://dx.doi.org/10.1016/j.bmcl.2007.03.080DOI Listing
May 2007

Sulfonamide-related conformational effects and their importance in structure-based design.

Bioorg Med Chem Lett 2007 May 16;17(10):2931-4. Epub 2007 Feb 16.

GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.

Structure-based design (SBD) is a challenging endeavour since even localised SAR can hardly ever be explained by the variation of just one dominating factor. Here, we present a rare example where structural information combined with ab initio calculations clearly indicate that the observed difference in biological activity is dominated by conformational effects. The learnings discussed are successfully put to the test and have the potential to be of general use as a qualitative guide in SBD efforts.
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http://dx.doi.org/10.1016/j.bmcl.2007.02.034DOI Listing
May 2007

Global analysis of proteins synthesized by Mycobacterium smegmatis provides direct evidence for physiological heterogeneity in stationary-phase cultures.

J Bacteriol 2005 Oct;187(19):6691-700

Division of Biology, Faculty of Life Sciences, Imperial College London, Sir Alexander Fleming Building, Imperial College Road, London SW7 2AZ, United Kingdom.

We have characterized the induction kinetics of approximately 1,700 proteins during entry into and survival in carbon-starved stationary phase by Mycobacterium smegmatis. Strikingly, among the patterns of expression observed were a group of proteins that were expressed in exponential-phase cultures and severely repressed in 48-h stationary-phase cultures (Spr or stationary-phase-repressed proteins) but were synthesized again at high levels in > or =128-day stationary-phase cultures (Spr(128) proteins). A number of Spr(128) proteins were identified, and they included the heat shock protein DnaK, the tricarboxylic acid cycle enzyme succinyl coenzyme A synthase, a FixA-like flavoprotein, a single-stranded DNA binding protein, and elongation factor Tu (EF-Tu). The identification of EF-Tu as an Spr(128) protein is significant, as ribosomal components are known to be expressed in a growth rate-dependent way. We interpreted these data in terms of a model whereby stationary-phase mycobacteria comprise populations of cells that differ in both their growth status and gene expression patterns. To investigate this further, we constructed gene fusions between the rpsL gene promoter (which heads the Mycobacterium smegmatis operon encoding the tuf gene encoding EF-Tu) or the rrnA promoter gene and an unstable variant of green fluorescent protein. While the majority of cells in old stationary-phase cultures had low levels of fluorescence and so rpsL expression, a small but consistently observed population of approximately 1 in 1,000 cells was highly fluorescent. This indicates that a small fraction of the cells was expressing rpsL at high levels, and we argue that this represents the growing subpopulation of cells in stationary-phase cultures.
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http://dx.doi.org/10.1128/JB.187.19.6691-6700.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251579PMC
October 2005

Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli.

Mol Microbiol 2003 Sep;49(5):1191-200

Computational and Structural Sciences, GlaxoSmithKline, Gunnells Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.

Two-component signal transduction (TCST) pathways are regulatory systems that are highly homologous throughout the bacterial kingdom. Their established role in virulence and absence in vertebrates has made TCST an attractive target for therapeutic intervention. However, such systems have yet to yield success in the development of novel antibiotics. CheY serves as a prototype for the analysis of response regulator function. The protein structure exhibits several conformations by both X-ray and nuclear magnetic resonance (NMR) analyses. Knowledge of which structures are relevant in vivo would be valuable in a rational drug design project. Our aim was to probe the in vivo conformation and ligand binding of CheY in Escherichia coli under resting conditions by in-cell NMR methods. CheY was selectively labelled with 15N by the control of growth and expression conditions. NMR spectra obtained in vivo demonstrated that the Mg2+ complex was the predominant form even though cells were resuspended in metal-free buffers and the intracellular free Mg2+ was low. In-cell NMR also confirmed the uptake and in vivo binding mode to CheY of small-molecular-weight compounds identified in vitro. This paper reports the first observation of the structure and interactions with a potential drug of a regulator protein in its native host in vivo using NMR spectroscopy.
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http://dx.doi.org/10.1046/j.1365-2958.2003.03628.xDOI Listing
September 2003