Biology subjects

Rasouli, A.

Publications and source records attributed to Rasouli, A..

3 recordsLinked to original sources

bioRxiv2022

Structure of C. elegans TMC-1 complex illuminates auditory mechanosensory transduction

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction (MT) channel. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding MT, the composition, structure and mechanism of the MT complex has remained elusive. Here we report the single particle cryo-EM structure of the native MT TMC-1 complex isolated from C. elegans. The 2-fold symmetric complex is composed of 2 copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits while the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of particles in addition harbors a single arrestin-like protein, ARRD-6, bound to a CALM-1 domain. Single- particle reconstructions and molecular dynamics simulations show how the MT complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

biochemistry↗

bioRxiv2022

Tuning aromatic contributions by site-specific encoding of fluorinated phenylalanine residues in bacterial and mammalian cells

The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a new group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools will enable deconstruction of biological mechanisms which utilize aromatic-pi interactions in structural and cellular contexts. Statement of SignificanceThe aromatic side-chains of phenylalanine, tyrosine, and tryptophan are crucial for protein function and pharmacology due to their hydrophobic and electrostatic contributions to catalytic centers and ligand-binding pockets. However, few experimental approaches can chemically assess the functional roles of aromatics in cellular environments. The accepted computational method for aromatic interrogation is via serial fluorination, which lacks an experimental correlate in bacterial or mammalian cell systems. We have identified a family of synthetases to encode multiple different types of fluorinated phenylalanine residues in E. coli and HEK cells via nonsense suppression. The efficiency of these synthetases is sufficient to support biochemical characterization and structural determination of proteins with site-specific incorporation of unnatural phenylalanine analogs.

biochemistry↗

bioRxiv2022

Protonation-dependent alternating access of a spinster transporter, an emerging family of broad-specificity efflux pumps

Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange. Furthermore, our resistance assays reveal substrate polyspecificity and HnSpns multidrug resistance (MDR) activity that underscore the previously unknown role of Spns proteins in MDR, beyond their activity in sphingolipid transport and signaling.

biophysics↗