Le, S. N. | Search bioRxiv

bioRxiv2019

Structural studies of thyroid peroxidase show the monomer interacting with autoantibodies in thyroid autoimmune disease

Thyroid peroxidase (TPO) is a critical membrane-bound enzyme involved in the biosynthesis of multiple thyroid hormones, and is a major autoantigen in autoimmune thyroid diseases such as Graves disease and Hashimotos thyroiditis. Here we report the biophysical and structural characterisation of two novel TPO constructs containing only the ectodomain of TPO and lacking the propeptide. Both constructs were enzymatically active and able to bind the patient-derived TR1.9 autoantibody. Analytical ultra-centrifugation data suggests that TPO can exist as both a monomer and a dimer. Combined with negative stain electron microscopy and molecular dynamics simulations, these data show that TR1.9 autoantibody preferentially binds the TPO monomer, revealing conformational changes that bring together previously disparate residues into a continuous epitope. In addition to providing plausible structural models of a TPO-autoantibody complex, this study provides validated TPO constructs that will facilitate further characterization, and advances our understanding of the structural, functional and antigenic characteristics of TPO, a molecule behind some of the most common autoimmune diseases.

biochemistry↗

bioRxiv2019

AgarFix: simple and accessible stabilization of challenging single-particle cryo-EM specimens through cross-linking in a matrix of agar

Cryogenic electron microscopy (cryo-EM) allows structure determination of macromolecular assemblies that have resisted other structural biology approaches because of their size and heterogeneity. These challenging multi-protein targets are typically susceptible to dissociation and/or denaturation upon cryo-EM grid preparation, and often require cross-linking prior to freezing. Several approaches for gentle on-column or in-tube crosslinking have been developed. On-column cross-linking is not widely applicable because of the poor separation properties of gel filtration techniques. In-tube crosslinking frequently causes sample aggregation and/or precipitation. Gradient-based cross-linking through the GraFix method is more robust, but very time-consuming and necessitates specialised expensive equipment. Furthermore, removal of the glycerol typically involves significant sample loss and may cause destabilization detrimental to the sample quality. Here, we introduce an alternative procedure: AgarFix (Agarose Fixation). The sample is embedded in an agarose matrix that keeps the molecules separated, thus preventing formation of aggregates upon cross-linking. Gentle cross-linking is accomplished by diffusion of the cross-linker into the agarose drop. The sample is recovered by diffusion or electroelution and can readily be used for cryo-EM specimen preparation. AgarFix requires minimal equipment and basic lab experience, making it widely accessible to the cryo-EM community.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC=\"FIGDIR/small/569087_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (15K):\norg.highwire.dtl.DTLVardef@72305borg.highwire.dtl.DTLVardef@c4c978org.highwire.dtl.DTLVardef@186f823org.highwire.dtl.DTLVardef@cfd16_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFragile protein complexes can be cross-linked while embedded in agarose\nC_LIO_LIThe agarose matrix prevents formation of aggregates upon cross-linking\nC_LIO_LICross-linking in agarose is easy, fast, and requires no expensive equipment\nC_LIO_LIThe sample can directly be used for negative stain or cryo-EM grid preparation\nC_LI

biochemistry↗