GAD65 conformational dynamics paper published in PNAS

Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis

Kass et al, PNAS, Early online edition June 9th, 2014

See also Monash Media releases:

‘Jekyll and Hyde’ protein linked to type 1 diabetes

The autoimmune cost of shape-shifting brain protein 

Open and closed dimers of GAD65 and the mechanism of dimer opening in holo → apo conversion

Abstract
The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5′-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5′-phosphate–binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

 

New papers published

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease.

Hargreaves CE, Grasso M, Hampe CS, Stenkova A, Atkinson S, Joshua GW, Wren BW, Buckle AM, Dunn-Walters D, Banga JP.

Molecular determinants of the substrate specificity of the complement initiating protease, C1r.

Wijeyewickrema LC, Yongqing T, Tran TP, Thompson PE, Viljoen JE, Coetzer TH, Duncan RC, Kass I, Buckle AM, Pike RN.

J Biol Chem. 2013 Apr 15. [Epub ahead of print]

Mechanism-based selection of a potent kallikrein-related peptidase 7 inhibitor from a versatile library based on the sunflower trypsin inhibitor SFTI-1.

de Veer SJ, Ukolova SS, Munro CA, Swedberg JE, Buckle AM, Harris JM.

Biopolymers. 2013 Mar 11. doi: 10.1002/peps.22231. [Epub ahead of print]

Structural characterization of the mechanism through which human glutamic acid decarboxylase auto-activates.

Langendorf CG, Tuck KL, Key TL, Fenalti G, Pike RN, Rosado CJ, Wong AS, Buckle AM, Law RH, Whisstock JC.

Biosci Rep. 2013 Jan 11;33(1):137-44. doi: 10.1042/BSR20120111.

New paper published

Characterisation of Peptide Microarrays for Studying Antibody-Antigen Binding Using Surface Plasmon Resonance Imagery. PLoS ONE 5(8): e12152. doi:10.1371/journal.pone.0012152

Claude Nogues, Hervé Leh, Christopher G. Langendorf, Ruby H. P. Law, Ashley M. Buckle#*, Malcolm Buckle#*

1 Dynamics of Macromolecular Complexes, Laboratoire de Biologie et Pharmacologie Appliquée, UMR 8113 du CNRS, Institut d’Alembert, Ecole Normale Supérieure de Cachan, Cachan, France

2 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

Background
Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules.

Methodology/Principal Findings
We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65) and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van’t Hoff type analyses to provide comparative ΔG, ΔS and ΔH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding.

Conclusions
The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen interaction where good structural, mechanistic and immunological data are available. Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods. Furthermore, our data indicate that SPRi is well suited to a multiplexed immunoassay using GAD65 proteins, and may be applicable to other biomarkers.

Four new papers published:

Swedberg JE, Nigon LV, Reid JC, de Veer SJ, Walpole CM, Stephens CR, Walsh TP, Takayama TK, Hooper JD, Clements JA, Buckle AM & Harris, JM.  (2009) Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4.  Chemistry & Biology, 16, 633-43. PubMed Link

Fischer K,Langendorf  CG, Irving  JA, Reynolds S, Willis C, Beckham S, Law RHP, Yang S, Bashtannyk-Puhalovich TA, McGowan S, Whisstock JC, PikeRN, KempDJ, and Buckle AM  (2009) Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues. J. Mol. Biol. 390(4):635-45. PubMed Link

Fenalti G and Buckle AM (2010) Structural Biology of the GAD Autoantigen. Autoimmunity Reviews 9(3):148-52. Epub 2009 May 22. PubMed Link

Arafat Y, Fenalti G, Whisstock JC, Mackay IR, Garcia de la Banda M, Rowley MJ and Buckle AM (2009) Structural Determinants of GAD Antigenicity. Molecular Immunology, 47(2-3):493-505. PubMed Link