Structural and thermodynamic analyses of the β-to-α transformation in RfaH reveal principles of fold-switching proteins

The two-domain protein RfaH, a paralog of the universally conserved NusG/Spt5 transcription factors, is regulated by autoinhibition coupled to the reversible conformational switch of its 60- residue C-terminal KOW domain between an α-hairpin and a β-barrel. In contrast, NusG/Spt5-KOW domains only occur in the β-barrel state. To understand the principles underlying the drastic fold switch in RfaH, we elucidated the thermodynamic stability and the structural dynamics of two RfaH- and four NusG/Spt5-KOW domains by combining biophysical and structural biology methods. We find that the RfaH-KOW β-barrel is thermodynamically less stable than that of most NusG/Spt5-KOWs and we show that it is in equilibrium with a globally unfolded species, which, strikingly, contains two helical regions that prime the transition towards the α-hairpin. Our results suggest that transiently structured elements in the unfolded form might drive the global folding transition in metamorphic proteins in general.

Distinct roles of the Na+ binding sites in the allosteric coupling mechanism of the glutamate transporter homolog, GltPh.

Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to GltPh. The binding sites for three Na+ ions (Na1-3) have been identified in GltPh but the specific roles of the individual Na+ sites in the binding process has not been elucidated. In this study, we developed assays to probe Na+ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na+ binding to the Na3 site is required for the NMDGT conformational switch while Na+ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na+ binding to both the Na1 and the Na3 sites. Based on our studies we also propose an alternate pathway for the coupled binding of Na+ and Asp.

Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA–based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

Salt leverages polyethylene terephthalate hydrolase (PETase) enzymatic activity via the predicted conformational switch

Plastic pollution spawned a global challenge caused by the environmental accumulation of polyethylene terephthalate (PET) plastics. Ongoing remediation efforts using microbial and engineered PET hydrolyzing enzymes (PETases) are hindered by slow depolymerization activities. Here, we report the optimized reaction conditions that leveraged the PETase hydrolase activity 2 to 3.8-fold in the presence of high NaCl concentrations. Molecular dynamics simulations (MDS) were applied to model salt-dependent conformational changes of the PETase enzyme bound to a 3-unit PET polymer. MDS demonstrated that PETase interaction with flanking polymer units exhibited a striking structural disparity at low and high salt concentrations. At low salt concentrations, flanking polymer units displayed significant bending. In contrast, flanking units extended at high salt concentrations, thus residues D206, H237, and S160 of the catalytic triad positioned in close vicinity of the scissile ester bond of the polymer substrate. The resulting high salt-specific enzyme/substrate geometry can potentially facilitate hydrolysis. We theorized that a salt-dependent conformational switch could attenuate the enzyme to a broad range of natural and artificial polymers consumed as carbon sources. Altogether, new knowledge may advance the engineering of PET hydrolase enzymes and benefit bioconversion programs.

A conformational switch controlling the toxicity of the prion protein

SummaryPrion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond (“H-latch”) altering the flexibility of the α2-α3 and β2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

Conformational plasticity of the ULK3 kinase domain

The human protein kinase ULK3 regulates the timing of membrane abscission, thus being involved in exosome budding and cytokinesis. Herein, we present the first high-resolution structures of the ULK3 kinase domain. Its unique features are explored against the background of other ULK kinases. An inhibitor fingerprint indicates that ULK3 is highly druggable and capable of adopting a wide range of conformations. In accordance with this, we describe a conformational switch between the active and an inactive ULK3 conformation, controlled by the properties of the attached small-molecule binder. Finally, we discuss a potential substrate-recognition mechanism of the full-length ULK3 protein.