Ständer Susanne H D, Reboul Cyril F, Le Sarah N, Williams Daniel E, Chandler Peter G, Costa Mauricio G S, Hoke David E, Jimma John D T, Fodor James, Fenalti Gustavo, Mannering Stuart I, Porebski Benjamin T, Schofield Peter, Christ Daniel, Buckle Malcolm, McGowan Sheena, Elmlund Dominika, Rand Kasper D, Buckle Ashley M
Protein Analysis Group, Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark.
Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
Nat Commun. 2025 Mar 7;16(1):2275. doi: 10.1038/s41467-025-57492-4.
The enzyme glutamate decarboxylase (GAD) produces the neurotransmitter GABA, using pyridoxal-5'-phosphate (PLP). GAD exists as two isoforms, GAD65 and GAD67. Only GAD65 acts as a major autoantigen, frequently implicated in type 1 diabetes and other autoimmune diseases. Here we characterize the structure and dynamics of GAD65 and its interaction with the autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11. Using hydrogen-deuterium exchange mass spectrometry (HDX), X-ray crystallography, cryo-electron microscopy, and computational approaches, we examine the conformational dynamics of apo- and holoGAD65 and the GAD65-autoantibody complex. HDX reveals local dynamics accompanying autoinactivation, with the catalytic loop promoting collective motions at the CTD-PLP domain interface. In the GAD65-b96.11 complex, heavy chain CDRs dominate the interaction, with a long CDRH3 bridging the GAD65 dimer via electrostatic interactions with the PEVKEKmotif. This bridging links structural elements controlling GAD65's conformational flexibility to its autoantigenicity. Thus, intrinsic dynamics, rather than sequence differences within epitopes, appear to be responsible for the contrasting autoantigenicities of GAD65 and GAD67. Our findings elucidate the structural and dynamic factors that govern the varying autoantibody reactivities of GAD65 and GAD67, offering a revised rationale for the autoimmune response to GAD65.
谷氨酸脱羧酶（GAD）利用磷酸吡哆醛（PLP）产生神经递质γ-氨基丁酸（GABA）。GAD以两种异构体形式存在，即GAD65和GAD67。只有GAD65作为主要自身抗原，经常与1型糖尿病和其他自身免疫性疾病有关。在此，我们对GAD65的结构和动力学及其与2型自身免疫性多内分泌综合征相关自身抗体b96.11的相互作用进行了表征。我们使用氢-氘交换质谱（HDX）、X射线晶体学、冷冻电子显微镜和计算方法，研究了无辅基和全酶形式的GAD65以及GAD65-自身抗体复合物的构象动力学。HDX揭示了伴随自身失活的局部动力学，催化环在CTD-PLP结构域界面促进集体运动。在GAD65-b96.11复合物中，重链互补决定区主导相互作用，长的CDRH3通过与PEVKEK基序的静电相互作用桥接GAD65二聚体。这种桥接将控制GAD65构象灵活性的结构元件与其自身抗原性联系起来。因此，内在动力学而非表位内的序列差异似乎是GAD65和GAD67自身抗原性差异的原因。我们的研究结果阐明了控制GAD65和GAD67不同自身抗体反应性的结构和动力学因素，为针对GAD65的自身免疫反应提供了修正的理论依据。
