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Ruby Law

Research areas:

Fibrinolytic system: This work is funded by NHMRC in collaboration with A/Professor Paul Coughlin and Dr Anita Horvath from the Australian Blood Disease Centre; and Drs Tom Caradoc-Davis and Nathan Cowieson from the Australian Synchrotron. Plasminogen is the zymogen form of plasmin which is the most formidable protease in the plasma. Plasmin is responsible for the removal of blood clots (fibrinolysis) in the circulating system in order to maintain the blood flow. As well as that, plasminogen can also bind to the surface of cells and become activated to plasmin, which breaks down the basement membrane and extra-cellular matrix hence promotes cell migration and, in the case of cancer, cell invasion. We crystallized and determined the crystal structure of plasminogen, and the results were recently published in Cell Reports (2012). The structure reveals how circulating plasminogen resists activation until it binds to a fibrin clot or target cell surface. It also explains how pathogens (such as Streptococcus) hijack this potent plasma protease during invasion. Our current focus is to understand the molecular interactions during plasminogen activation and plasmin inhibitions, especially in disease conditions such as thrombosis and uncontrolled bleeding. We employ various techniques including mutagenesis, characterization of enzyme activities, protein complex formation, X-ray crystallography and small angle X-ray scattering.

http://www.cell.com/cell-reports/abstract/S2211-1247%2812%2900069-1

Structure and regulation of function of human glutamic decarboxylase (GAD): Gamma aminobutyric acid (GABA) produced by GAD is the most abundant neurotransmitter inhibitor in the CNS and is critical for the control of movements, and neuroplasticity during development, learning and recovery from brain damage. Neurological conditions, such as anxiety, autism and post-traumatic stress disorder, are closely related to the imbalance of GABA homeostasis. This project is a continuation and expansion of our previous work on GAD production project for diagnostic and immunological tolerance studies. We published the crystal structures of both GAD65 and GAD67 isoforms in Nature Structure and Molecular Biology (2007). We observed that the mechanism through which GABA produced in mammals is regulated by a dynamic catalytic loop. The two isoforms of GAD cooperate to meet different physiological circumstances; GAD67 is a housekeeping enzyme which maintains the basal level of GABA in the CNS and stably binds to the cofactor pyridoxal 5’ phosphate (as a holo-enzyme), whilst GAD65 switches between the apo and holo-form through a process called autoinactivation. The current project studies firstly, the structural motifs involved in the autoinactivation and secondly, small molecules which can modulate the rate of GAD activity. These studies involve measuring enzyme activity and using X-ray crystallography; with our outstanding research assistant turned PhD student Chris Langendorf being the key contributor to this project. Our aim is to comprehensively understand the process of auto-inactivation and allosteric regulation of GAD, with the long term aim of developing therapeutic treatments for GAD neurological diseases through the modulation of GAD activities.

http://www.nature.com/nsmb/journal/v14/n4/covers/index.html

Transmembrane pore formation in perforin: Perforin plays a critical role in the immune homeostasis and surveillance against viral infection and cancer cells and deficiency is associated with impaired cytotoxic T-lymphocyte function. Perforin is a pore-forming MACPF protein (Membrane Attack Complex and Perforin-like) stored as a soluble conformer in the cytoplasmic granules of natural killer cells and cytotoxic T-lymphocytes. Upon conjugation with target cells, perforin oligomerises and adopts a new conformation during pore formation. The perforin trans-membrane pores mediate the delivery of pro-apoptotic granzymes which induce cytolysis. In collaboration with Professor Joe Trapani and Dr Ilia Voskoboinik from Peter MacCallum Cancer Research Institute, we characterized the X-ray crystal structure of the monomeric perforin. At the same time Professor Helen Saibil’s group from Birkbeck College in London determined the perforin monomer and pore structure by cryo-electron microscopy. Together we were able to model how perforin assembles on the surface of the target cell upon formation of transmembrane pores; these results were published in Nature (2010). This publication is a very rewarding outcome following our previous paper (in collaboration with Dr Michelle Dunstone) published in Science (2007) describing for the first time that the MACPF proteins, including those found in the mammalian immunity defense system, adopt the same fold as the bacterial cholesterol dependent cytolysins. Our current objective is to understand at the molecular level how perforin binds to the cell membrane and forms pores on the surface of the target cells.

http://www.ncbi.nlm.nih.gov/pubmed/21037563

http://www.ncbi.nlm.nih.gov/pubmed/17872444

Publications:

McGowan S, Buckle AM, Mitchell MS, Hoopes JT, Gallagher DT, Heselpoth RD, Shen Y, Reboul CF, Law RHP, Fischetti WA, Whisstock JC, and Nelson DC (2012)¬ X-ray crystal structure of the streptococcal specific phage lysin PlyC. Proceedings of the national Academy of the United States of America, in press.

Law RHP*, Caradoc-Davies TT, Cowieson N, Horvath AJ, Quek AJ, Encarnacao JA, Steer D, Cowan A, Zhang Q, Lu BGC, Pike RN, Smith AI, Coughlin PB, Whisstock JC (2012) The X-ray Crystal Structure of Full-Length Human Plasminogen. Cell Reports 1(3):185-190.

Lu BG, Sofian T, Law RH, Coughlin PB, Horvath AJ (2011) The contribution of conserved lysine residues in the {alpha}2-antiplasmin C-terminus to plasmin binding and inhibition. J Biol Chem. 286 (28):24544-24552.

Law RHP*, Lukoyanova N, Voskoboinik I, Caradoc-Davies TT, Baran K, Dunstone MA, D’Angelo ME, Orlova EV, Coulibaly F, Verschoor S, Browne KA, Ciccone A, Kuiper MJ, Bird PI, Trapani JA, Sailil HR, Whisstock JC (2010) The Structural basis for membrane binding and pore formation by lymphocyte perforin. Nature 468(7322):447-51.

Kennan RM, Wong W, Dhungyel O, Han X, Wong D, Parker D, Rosado CJ, Law RHP, McGowan S, Reeve S, Levina V, Powers GA, Pike RN, Bottomley SP, Smith AI, Marsh I, Whittington RJ, Whisstock JC, Porter CJ, Rood JL (2010). The subtilisin-like proteases APrV2 is required for virulence and uses a novel disulphide-tethered exocite to bind substrate. PLoS Pathog 6(11):e1001210.

Kondos SC, Hatfaludi T, Voskoboinik I, Trapani JA, Law RH, Whisstock JC, Dunstone MA(2010) The structure and function of mammalian membrane-attack complex/perforin-like proteins. Tissue Antigens 76, 341-51.

Nogues C, Leh H, Langendorf CG, Law RH, Buckle AM, Buckle M (2010) Characterisation of peptide microarrays for studying antibody-antigen binding using surface plasmon resonance imagery. PLoS One 13(8):e12152.

Langendorf CG, Key TL, Fenalti G, Kan WT, Buckle AM, Caradoc-Davies T, Tuck KL, Whisstock JC, Law RH** (2010) The X-ray crystal structure of Escherichia coli succinic semialdehyde dehydrogenase; structural insights into NADP+/enzyme interactions. PLoS One 18(2):e9280.

Song J, Tan H, Mahmood K, Law RH, Buckle AM, Webb GI, Akutsu T, Whisstock JC (2009) Prodepth: predict residue depth by support vector regression approach from protein sequences only. PLoS One 17(9):e7072.

Fischer K, Langendorf CG, Irving JA, Reynolds S, Willis C, Beckham S, Law RH, Yang S, Bashtannyk-Puhalovich TA, McGowan S, Whisstock JC, Pike RN, Kemp DJ, Buckle AM (2009). Structural mechanisms of inactivation in scabies mite serine protease paralogues. J Mol Biol Jul 24;390(4):635-45.

Beckham SA, Piedrafita D, PhillipsCI, Samarawickrema N, Law RHP, Smooker PM, Quinsey NS, Irving JA, Greenwood D, Verhelst SHL, Bogyo M, Turk B, Coetzer TH, Wijeyewickrema LC, Spithill TW and Pike RN (2009) A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites Journal of Biochemistry and cell Biology 41:1601-1622.

Kaiserman D, Buckle AM, Van Damme P, Irving JA, Law RHP, Matthews AY, Bashtannyk-Puhalovich T, Langendorf C, Thompson P, Vandekerckhove J, Gevaert K, Whisstock JC, Bird P (2009) Structure of granzyme C reveals a novel mechanism of protease auto-inhibition Proceedings of the national Academy of the United States of America 106(14):5587-5592.

Estuningsih E, Spithill T, Raadsma H, Law R, Adiwinata G, Meeusen E, Piedrafita D (2009) Development and Application of A Faecal Antigen Diagnostic Sandwich ELISA For Estimating Prevalence of Fasciola gigantica In Cattle In Central Java, Indonesia Journal of Parasitology 95(2):450-455.

Fenalti G, Hampe CS, Arafat Y, Law RH, Banga JP, Mackay IR, Whisstock JC, Buckle AM, Rowley MJ (2008) COOH-terminal clustering of autoantibody and T-cell determinants on the structure of GAD65 provide insights into the molecular basis of autoreactivity Diabetes 57(5):1293-301.

Rosado CJ, Kondos S, Bull TE, Kuiper MJ, Law RH, Buckle AM, Voskoboinik I, Bird PI, Trapani JA, Whisstock JC, Dunstone MA (2008) The MACPF/CDC family of pore-forming toxins Cell Microbiology 10(9):1765-74.

Zhang Q, Law RHP, Bottomley SP, Whisstock JC, Buckle AM (2008) A structural Basis for Loop C-Sheet Polymerization in Serpins Journal of Molecular Biology 376(5):1348-59.

Law RHP*, Sofian T, Kan WT, Horvath AJ, Hitchen CR, Langendorf CG, Buckle AM, Whisstock JC, Coughlin PB (2008) The X-ray crystal structure of the fibrinolysis inhibitor α2-antiplasmin Blood 111(4):2049-52.

Law RHP*, Rosado CJ, Buckle AM, Butcher RE, Kan WT, Bird CH, Ung K, Browne KA, Baran K, Bashtannyk-Puhalovich TA, Faux NG, Wong W, Porter CJ, Pike RN, Ellisdon AM, Pearce MC, Bottomley SP, Emsley J, Smith AI, Rossjohn J, Hartland EL,Voskoboinik I, Trapani JA, Bird PI, Dunstone MA, Whisstock JC (2007) A common fold mediates vertebrate defense and bacterial attack Science 317(5844):1548-51.

Law RHP*, Zhang Q, Buckle AM, Pearce MC, Cabrita LD, Lloyd GJ, Irving JA, Smith AI, Ruzyla K, Rossjohn J, Bottomley SP, Whisstock JC (2007) The N terminus of the serpin, tengpin, functions to trap the metastable native state EMBO Reports 8(7):658-63.

Law RHP*, Fenalti G, Buckle AM, Langendorf C, Tuck K, Rosado CJ, Faux NG, Mahmood K, Hampe CS, Banga JP, Wilce M, Schmidberger J, Rossjohn J, El-Kabbani O, Pike RN, Smith AI, Mackay IR, Rowley MJ, Whisstock JC (2007) GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop Nature Structural and Molecular Biology 14(4):280-6.

Zhang Q, Law R, Buckle AM, Cabrita L, McGowan S, Irving JA, Faux NG, Lesk AM, Bottomley SP and Whisstock JC (2006) Serpins in Prokaryotes in Molecular and Cellular Aspects of the Serpinopathies and Disorders in Serpin Activity (Silverman G and Lomas DA ed), World Scientific Publishers, USA, pp 131-162.

Law RHP*, Hennebry SC, Richardson SJ, Buckle AM, Whisstock JC (2006) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase Journal of Molecular Biology 359(5):1389-99.

Law RHP, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong W, Rosado CJ, Langendorf CG, Pike RN, Bird PI, Whisstock JC (2006) An overview of the serpin superfamily Genome Biology 7(5):216.

Amin AA, Faux NG, Fenalti G, Williams G, Bernadou A, Daglish B, Keefe K, Middleton S, Rae J, Tetis K, Law RHP, Fulton KF, Rossjohn J, Whisstock JC, Buckle AM (2006) Managing and mining protein crystallization data Proteins 62(1):4-7.

Horvath AJ, Irving JA, Rossjohn J, Law RHP, Bottomley SP, Quinsey NS, Pike RN, Coughlin PB, Whisstock JC (2005) The murine orthologue of human antichymotrypsin: a structural paradigm for clade A3 serpins Journal of Biological Chemistry 280(52):43168-78.

Law RHP*, Irving JA, Buckle AM, Ruzyla K, Buzza M, Bashtannyk-Puhalovich TA, Beddoe T.C, Nguyen K, Worrall DM, Bottomley SP, Bird PI, Rossjohn J, Whisstock JC (2005) The high resolution crystal structure of the human tumor suppressor maspin reveals a novel conformational switch in the G-helix Journal of Biological Chemistry 280(23):22356-64.