Biology subjects

Wright, P. E.

Publications and source records attributed to Wright, P. E..

4 recordsLinked to original sources

bioRxiv2025

Linker Length and Composition within Disordered Binding Motifs modulates the Avidity and Reversibility of a Multivalent Protein Interaction Switch

Intrinsically disordered proteins that mediate the cellular transcriptional response to hypoxia play important roles in turning on and turning off oxygen stress genes. In particular, the feedback inhibitor CITED2 operates a unidirectional switch that efficiently terminates the hypoxic response by displacing the C-terminal activation domain of the hypoxia-inducible factor HIF-1 from its complex with the TAZ1 domain of the transcriptional coactivators CBP and p300. Unidirectionality of the switch arises from subtle allosteric conformational changes in TAZ1 and from differences in the strength of thermodynamic coupling between the TAZ1-binding motifs in the multivalent HIF-1 and CITED2 activation domains. To investigate the role of binding cooperativity, we mutated a linker sequence in the HIF-1 activation domain to enhance or reduce the thermodynamic coupling between its TAZ1-binding motifs. An efficient native-gel assay shows that certain linker mutations enhance the affinity of HIF-1 for TAZ1, and fluorescence anisotropy competition and NMR measurements show that these mutants can compete with CITED2 for TAZ1 more effectively than wild-type HIF-1. The wide range of mutants, which include insertion, deletion, replacement and scrambling of residues in the linker, provide insights into the molecular basis for the exquisite tuning of the hypoxic switch: the TAZ1 affinity and consequent CITED2 competition enhancement depends both on the flexibility of the linker sequence (particularly the presence of glycine residues) and the unfavorable electrostatic interactions of a highly conserved arginine side chain in the center of the linker with an electropositive surface of TAZ1.

biophysics↗

bioRxiv2024

Switchable client specificity in a dual functional chaperone coordinates light harvesting complex biogenesis

The proper assembly of light harvesting complexes (LHCs) is critical for photosynthesis and requires the biogenesis of light-harvesting chlorophyll a,b-binding proteins (LHCPs) to be coordinated with the biosynthesis of chlorophylls (Chl). The mechanism underlying this coordination is not well understood. Here we show that a conserved molecular chaperone in chloroplasts, cpSRP43, provides a molecular thermostat that helps maintain this coordination. cpSRP43 undergoes a conformational rearrangement between a well-folded closed state and a partially disordered open state. Closed cpSRP43 is dedicated to the de novo biogenesis of LHCPs, whereas open cpSRP43 protects multiple Chl biosynthesis enzymes from heat-induced destabilization. Rising temperature shifts cpSRP43 to the open state and thus enables it to protect Chl biosynthesis enzymes that are heat-destabilized. Our results reveal the molecular basis of a post-translational mechanism for the thermo-adaptation of LHC biogenesis. They also demonstrate how an ATP-independent chaperone uses conformational dynamics to switch its activity and client selectivity, thereby adapting to different proteostatic demands under shifting environmental conditions. TeaserA thermo-switchable molecular chaperone helps coordinate light harvesting complex assembly during photosynthesis.

biochemistry↗

bioRxiv2024

Aggregation of Transthyretin by Fluid Agitation

The transthyretin (TTR) tetramer, assembled as a dimer of dimers, transports thyroxine and retinol binding protein in blood plasma and cerebrospinal fluid. Aggregation of wild type or pathogenic variant TTR leads to transthyretin amyloidosis (ATTR), which is associated with neurodegenerative and cardiac disease. The trigger for TTR aggregation under physiological conditions is unknown. The tetramer is extremely stable at neutral pH, but aggregation via tetramer dissociation and monomer misfolding can be induced in vitro by lowering the pH. To elucidate factors that may cause TTR aggregation at neutral pH, we examined the effect of shear forces such as arise from fluid flow in the vascular system. Fluid shear forces were generated by rapidly stirring TTR solutions in conical microcentrifuge tubes. Under agitation, TTR formed {beta}-rich aggregates and fibrils at a rate that was dependent upon protein concentration. The lag time before the onset of agitation-induced aggregation increases as the total TTR concentration is increased, consistent with a mechanism in which the tetramer first dissociates to form monomer that either partially unfolds to enter the aggregation pathway or reassociates to form tetramer. NMR spectra recorded at various time points during the lag phase revealed growth of an aggregation-prone intermediate trapped as a dynamically perturbed tetramer. Enhanced conformational fluctuations in the weak dimer-dimer interface suggests loosening of critical inter-subunit contacts which likely destabilizes the agitated tetramer and predisposes it towards dissociation. These studies provide new insights into the mechanism of aggregation of wild type human TTR under near physiological conditions.

biophysics↗

bioRxiv2023

Probing the dissociation pathway of a kinetically labile transthyretin mutant

Aggregation of transthyretin (TTR) is associated with devastating TTR amyloid disease. Amyloidosis begins with dissociation of the native tetramer to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the dissociation and reassembly of TTR at neutral pH remains poorly understood, due to the low population of intermediates. We use NMR studies with a highly sensitive 19F probe that allows deconvolution of relative populations of a destabilized A25T mutant at concentrations as low as 2 {micro}M. The A25T mutation, located at the weak dimer interface, perturbs both the weak and strong dimer interfaces. A tetramer-dimer-monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. All thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by vant Hoff and 19F NMR line-shape analysis. The conversion from T to T*, the slowest first-order kinetic step, shows anti-Arrhenius behavior. The 19F and methyl chemical shifts of probes close to the strong dimer interface in the dimer and T* species are degenerate, implicating interfacial perturbation as a common structural feature of these intermediate species. Molecular dynamics (MD) simulations further suggest more frequent F87 ring flipping on the nanoscale timescale in the A25T dimer than in the tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

biophysics↗