Biology subjects

Assafa, T. E.

Publications and source records attributed to Assafa, T. E..

2 recordsLinked to original sources

bioRxiv2026

Spin-labeling studies implicate a highly dynamic active state for transducin-bound phosphodiesterase-6 in vertebrate phototransduction

In vertebrate phototransduction, the G protein-coupled receptor rhodopsin activates the -subunit of transducin (GT), which, upon binding the {gamma}-subunits of phosphodiesterase-6 (PDE6), stimulates cGMP hydrolysis. We reported a cryoEM structure for a complex containing two constitutively active GT (GT*) subunits coupled by a bivalent antibody bound to PDE6 that demonstrated a striking displacement of both PDE{gamma} subunits from the PDE/PDE{beta} catalytic sites and suggested an alternating-site mechanism for PDE6 activation. Here, we use site-directed spin labeling (SDSL) and double electron-electron resonance spectroscopy (DEER) to probe PDE6 conformational changes upon GT* binding. PDE{gamma} spin-labelled on Cys68 and Ile64Cys demonstrate that PDE{gamma} have highly flexible C-termini that transiently bind to the PDE/PDE{beta} heterodimer. Binding of GT* to PDE6 with the inhibitor udenafil occupying its catalytic sites alters the positions of the PDE{gamma} subunits in agreement with the changes shown in the cryoEM structure for this complex, whereas coupling the GT* subunits to the bivalent antibody does not affect the DEER distributions observed for PDE6 bound to GT*. However, binding of the slow hydrolyzing 8-Br-cGMP substrate in the presence of GT* causes a dramatic increase in the separation and spread of the spin-labelled PDE{gamma} subunits, thereby revealing a previously unobserved conformation of PDE6 associated with catalysis, which is further supported by small angle X-ray scattering (SAXS) analysis. These studies indicate that whereas inhibitors trap GT*-PDE6 complexes in an inactive state as represented by the cryoEM structure, the binding of both substrate and GT* produces a dynamic active state consistent with an alternating-site mechanism.

biochemistry↗

bioRxiv2024

Multi-state kinetics of the syringe-like injection mechanism of Tc toxins

Tc toxins are virulence factors of many insects and human pathogenic bacteria. They attach as soluble prepores to receptors on host cells and following acidification in the late endosome, perforate the cell membrane like a syringe to translocate toxic enzymes into the host cell through their pore-forming channel. Although this complex transformation has been structurally well studied, the functional aspects of this large-scale rearrangement, such as the reaction pathway with possible intermediate states and the resulting temporal evolution have remained elusive. Here, we used an integrated biophysical approach to monitor the prepore-to-pore transition and found that it takes [~]28 h when induced by high pH in the absence of other factors. In the presence of liposomes, an increasingly high pH or receptors, such as heparin or Vsg, the probability to transform prepores to pores increases by a factor of up to 4. This effect can also be mimicked by biotinylation or site-directed mutagenesis of the shell, demonstrating that shell destabilization is a crucial step in prepore-to-pore transition. We show that shell opening is a heterogeneous process with transition times ranging from 60 ms to 1.6 s and resolve three sequential intermediate states: an initial transient intermediate during shell destabilization, a first stable intermediate where the receptor-binding domains on the shell rearrange and a second stable intermediate with an open shell. In contrast, the ejection of the pore-forming channel from the open shell is highly cooperative with a transition time of < 60 ms. This detailed knowledge of the Tc toxin mechanism of action, even in the absence of receptors, is important for the future application of Tc toxins as biomedical devices or biopesticides.

biochemistry↗