Biology subjects

Buckle, M.

Publications and source records attributed to Buckle, M..

4 recordsLinked to original sources

bioRxiv2024

Structure and dynamics of the autoantigen GAD65 in complex with the human autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11

The enzyme glutamate decarboxylase (GAD) produces the neurotransmitter GABA, using pyridoxal-5-phosphate. GAD exists as two isoforms, GAD65 and GAD67. Only GAD65 acts as a major autoantigen, with its autoantibodies frequently found 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. Combining hydrogen-deuterium exchange mass spectrometry (HDX), X-ray crystallography, cryo-electron microscopy and computational approaches, we dissect the conformational dynamics of the inactive apo- and the active holo-forms of GAD65, as well as the structure of the GAD65-autoantibody complex. HDX reveals the time-resolved, local dynamics that accompany autoinactivation, with the catalytic loop playing a key role in promoting collective dynamics at the interface between CTD and PLP domains. In the GAD65-b96.11 complex, heavy chain CDRs dominate the interaction, with the relatively long CDRH3 at the interface centre and uniquely bridging the GAD65 dimer via extensive electrostatic interactions with the 260PEVKEK265 motif. The autoantibody bridges structural elements on GAD65 that contribute to conformational change in GAD65, thus connecting the unique and intrinsic conformational flexibility that governs the autoinactivation mechanism of the enzyme to its autoantigenicity. The intrinsic dynamics, rather than sequence differences within epitopes, appear to be responsible for the contrasting autoantigenicities of GAD65 and GAD67. Our data thus reveal insights into the structural and dynamic differences between GAD65 and GAD67 that dictate their contrasting autoantibody reactivities, provide a new structural rationalisation for the nature of the autoimmune response to GAD65, and may have broader implications for antigenicity in general.

biochemistry↗

bioRxiv2020

Mutational and biophysical robustness in a pre-stabilized monobody

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyper-stable monobody derivative with diagnostic and therapeutic potential. Pre-stabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain towards biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerisation. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the pre-stabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 {degrees}C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a pre-stabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics, and is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

biochemistry↗

bioRxiv2020

Nucleosome assembly and disassembly pathways.

Nucleosome assembly and disassembly play a central role in the regulation of gene expression. Here we use PhAST (Photochemical Analysis of Structural Transitions) to monitor at the base pair level, structural alterations induced all along DNA upon histone binding or release. By offering the first consistent, detailed comparison of nucleosome assembly and disassembly in vitro, we are able to reveal similarities and differences between the two processes. We identify multiple intermediate states characterised by specific PhAST signatures; revealing a complexity that goes beyond the known sequential events involving (H3-H4)2 tetramer and H2A-H2B heterodimers. Such signatures localise and quantify the extent of the asymmetry of DNA/histone interactions with respect to the nucleosome dyad. This asymmetry is therefore defined by the localisation and amplitude of the signals. The localisation of the signal is consistent between assembly and disassembly and dictated by the DNA sequence. However, the amplitude component of this asymmetry not only evolves during the assembly and disassembly but does so differently between the two processes. Understanding the regulation of gene expression requires a complete knowledge of nucleosome dynamics. Our unexpected observation of differences between assembly and disassembly opens up new avenues to define the role of the DNA sequence in these processes. Overall, we provide new insights into how the intrinsic properties of DNA are integrated into a holistic mechanism that controls chromatin structure. Statement of SignificanceThis manuscript addresses the question of nucleosome dissociation compares with association. We used PhAST which is a non-intrusive photochemical technique to follow nucleosome dynamics at base pair resolution. We observed structural asymmetry during nucleosome turnover. We also showed for the first time that the process of nucleosome dissociation is not a reversal of association. This asymmetry favours intermediate states involved in chromatin organisation suggesting novel models for the role of nucleosome turnover in the epigenetic regulation of gene expression.

molecular biology↗

bioRxiv2018

Structural Capacitance in Protein Evolution and Human Diseases

Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism, that we term structural capacitance, which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opoposed to exclusively canonical Darwinian mechanisms. Our results implicate the elements of protein microstructure generated by this distinct mechanism in the pathogenesis of a wide variety of human diseases. The benefits of rapidly furnishing the potential for evolutionary change conferred by structural capacitance are consequently counterbalanced by this accompanying risk, with the extent of this determined by the host immune system. The phenomenon of structural capacitance has implications ranging from the ancestral diversification of protein folds to the engineering of synthetic proteins with enhanced evolvability.

biochemistry↗