3 research outputs found
The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2Ī², Are Crucial to Autoantibody Binding in Type 1 Diabetes
Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2Ī² also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2Ī² resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2Ī² PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions
Design and Nuclear Magnetic Resonance (NMR) Structure Determination of the Second Extracellular Immunoglobulin Tyrosine Kinase A (TrkAIg2) Domain Construct for Binding Site Elucidation in Drug Discovery
The tyrosine kinase A (TrkA) receptor
is a validated therapeutic
intervention point for a wide range of conditions. TrkA activation
by nerve growth factor (NGF) binding the second extracellular immunoglobulin
(TrkAIg2) domain triggers intracellular signaling cascades. In the
periphery, this promotes the pain phenotype and, in the brain, cell
survival or differentiation. Reproducible structural information and
detailed validation of proteināligand interactions aid drug
discovery. However, the isolated TrkAIg2 domain crystallizes as a Ī²-strand-swapped
dimer in the absence of NGF, occluding the binding surface. Here we
report the design and structural validation by nuclear magnetic resonance
spectroscopy of the first stable, biologically active construct of
the TrkAIg2 domain for binding site confirmation. Our structure closely
mimics the wild-type fold of TrkAIg2 in complex with NGF (1WWW.pdb), and the <sup>1</sup>Hā<sup>15</sup>N correlation spectra confirm that both
NGF and a competing small molecule interact at the known binding interface
in solution