Biology subjects

Sigworth, F. J.

Publications and source records attributed to Sigworth, F. J..

4 recordsLinked to original sources

bioRxiv2026

SCN- induces prestin contraction through reduced intermonomer angles and a modified transporter like movement of each monomer.

Prestin is the membrane based molecular motor responsible for outer hair cell electromotility that expands and contracts in response to transmembrane voltage. How voltage brings these about is unknown but cryoEM structures in the presence of ligands has led to the extrapolation of structures with the transporter cycle and its expansion contraction coupling. We find prestin to adopt novel increasingly contracted states that are brought about by a reduced intermonomer angles in the obligate dimers and a transmembrane contraction of each monomer at its cytoplasmic half in the presence of SCN-. These changes are associated with a loss in the intermonomer beta sheet at its N terminus. There is a hyperpolarizing shift in Vh along with increased affinity for SCN-. SCN- binding at the internal binding site likely cause the structural changes since they are reversed by SCN- displacement by salicylate.

physiology↗

bioRxiv2023

Cryo-EM structures of Kv1.2 potassium channels, conducting and non-conducting

We present near-atomic-resolution cryo-EM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 [A], 2.5 [A], 3.2 [A], and 2.9[A]. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2-2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin -Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.

biophysics↗

bioRxiv2023

REPIC -- an ensemble learning methodology for cryo-EM particle picking

Cryo-EM (cryogenic electron microscopy) particle identification from micrographs (i.e., picking) is challenging due to the low signal-to-noise ratio and lack of ground truth for particle locations. Moreover, current computational methods ("pickers") identify different particle sets, complicating the selection of the best-suited picker for a protein of interest. Here, we present REPIC, an ensemble learning methodology that uses multiple pickers to find consensus particles. REPIC identifies consensus particles by framing its task as a graph problem and using integer linear programming to select particles. REPIC picks high-quality particles when the best picker is not known a priori and for known difficult-to-pick particles (e.g., TRPV1). Reconstructions using consensus particles achieve resolutions comparable to those from particles picked by experts, without the need for downstream particle filtering. Overall, our results show REPIC requires minimal (often no) manual picking and significantly reduces the burden on cryo-EM users for picker selection and particle picking. Availabilityhttps://github.com/ccameron/REPIC

bioinformatics↗

bioRxiv2022

Structural insights into sodium/iodide symporter (NIS) substrate binding and specificity

The sodium/iodide symporter (NIS) is the essential plasma membrane protein that mediates active iodide (I-) transport into the thyroid gland, the first step in the biosynthesis of the thyroid hormones--the master regulators of intermediary metabolism. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. For nearly 50 years before its molecular identification, NIS was already the molecule at the center of the single most effective internal radiation cancer therapy ever devised: radioiodide (131I-) treatment for thyroid cancer. Mutations in NIS cause congenital hypothyroidism, which must be treated immediately after birth to prevent stunted growth and cognitive deficiency. To date, the structure of NIS has been unknown. Here, we report three structures of rat NIS, determined by single-particle cryo-electron microscopy (cryo-EM): one with no substrates bound, one with 2 Na+ and 1 I- bound, and one with 1 Na+ and the oxyanion perrhenate bound. Structural analyses, functional characterization, and computational studies reveal the substrate binding sites and residues key for transport activity. Our results yield insights into how NIS selects, couples, and translocates anions--thereby establishing a framework for understanding NIS function--and into how it transports different substrates with different stoichiometries and releases substrates from its substrate-binding cavity into the cytosol.

biophysics↗