Biology subjects

Porebski, B. T.

Publications and source records attributed to Porebski, B. T..

6 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↗

bioRxiv2021

Emergence of function from single RNA sequences by Darwinian evolution

The spontaneous emergence of function from pools of random sequence RNA is widely considered an important transition in the origin of life. However, the plausibility of this hypothetical process and the number of productive evolutionary trajectories in sequence space are unknown. Here we demonstrate that function can arise starting from a single RNA sequence by an iterative process of mutation and selection. Specifically, we describe the discovery of both specific ATP or GTP aptamers - with micromolar affinity for their nucleotide ligand - starting each from a single, homopolymeric poly-A sequence flanked by conserved primer binding sites. Our results indicate that the ab initio presence of large, diverse random sequence pools is not a prerequisite for the emergence of functional RNAs and that the process of Darwinian evolution has the capacity to generate function even from single, largely unstructured RNA sequences with minimal molecular and informational complexity.

synthetic biology↗

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↗

bioRxiv2019

Phospho-tuning immunity through Denisovan, modern human, and mouse TNFAIP3 gene variants

Resisting or tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here, genomic analyses of anatomically modern humans, extinct Denisovan hominins, and mice revealed a series of missense variants in the immune response inhibitor A20 (encoded by TNFAIP3), substituting non-catalytic residues of the ubiquitin protease domain to diminish I{kappa}B-dependent phosphorylation and activation of A20. Two A20 variants with partial phosphorylation deficits appeared beneficial: one originating in Denisovans and introgressed in modern humans throughout Oceania, and another in a mouse strain resistant to Coxsackievirus. By contrast, a variant with 95% loss of phosphorylation caused spontaneous inflammatory disease in humans and mice. Analysis of the partial phosphorylation variant in mice revealed diminished tolerance of bacterial lipopolysaccharide or to poxvirus inoculation as trade-offs for enhanced immunity.\n\nOne Sentence SummaryModern and ancient variants reveal a genetically tunable element for balancing immunity and microbial tolerance.

immunology↗

bioRxiv2018

Reactive Centre Loop Dynamics and Serpin Specificity

Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as a1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from 1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.

biochemistry↗

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↗