Biology subjects

Tomasiak, T. M.

Publications and source records attributed to Tomasiak, T. M..

7 recordsLinked to original sources

bioRxiv2026

Structural basis of caspofungin inhibition of Nakaseomyces glabratus Fks1

The Fks1 catalytic subunit of fungal glucan synthase crosslinks UDP-glucose into {beta}-1,3-D-glucan in the fungal cell wall and is a target for the echinocandin class of antifungal drugs. Here, we present the resting state of Fks1 from Nakaseomyces glabratus (NgFks1) in both apo and caspofungin-bound states. Caspofungin binds the extracellular glucan exit portal of Fks1 adjacent to the product {beta}-1,3-D-glucan in a position similar to that reported for S. cerevisiae Fks1. Unlike S. cerevisiae Fks1, no Rho1 binding is present, but the glucan channel opening is in an open-like state at the resistance hotspot regions. However, the lower gate is closed, suggesting glucan is trapped from a previous round of catalysis. Our results show that caspofungin can bind in a mode in the absence of Rho1 in N. glabratus and point to possible species-specific differences in substrate translocation, inhibition, and resistance mechanisms.

biochemistry↗

bioRxiv2024

Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation

Many ATP-binding cassette (ABC) transporters are regulated by phosphorylation on long and disordered loops which present a challenge to visualize with structural methods. We have trapped an activated state of the regulatory domain (R-domain) of Yeast Cadmium Factor 1 (Ycf1) by enzymatically enriching the phosphorylated state. A 3.2 [A] cryo-EM structure reveals an R-domain structure with four phosphorylated residues and a position for the entire R-domain. The structure reveals key R-domain interactions including a bridging interaction between NBD1 and NBD2 as well as an interaction with the R-insertion, another regulatory region. We systematically probe these interactions with a linker substitution strategy along the R-domain and find a close match with these interactions and survival under Ycf1-dependent growth conditions. We propose a model where four overlapping phosphorylation sites bridge several regions of Ycf1 to engage in a transport-competent state.

biochemistry↗

bioRxiv2024

Structural Basis of Glutathione Recognition by the Yeast Cadmium Factor 1

Transporters from the ABCC family have an essential role in detoxifying electrophilic compounds including metals, drugs, and lipids, often through conjugation with glutathione complexes. The Yeast Cadmium Factor 1 (Ycf1) transports glutathione alone as well as glutathione conjugated to toxic heavy metals including Cd2+, Hg2+, and As3+. To understand the complicated selectivity and promiscuity of heavy metal substrate binding, we determined the cryo-EM structure of Ycf1 bound to the substrate, oxidized glutathione. We systematically tested binding determinants with cellular survival assays against cadmium to determine how the substrate site accommodates differentsized metal complexes. We identify a "flex-pocket" for substrate binding that binds glutathione complexes asymmetrically and flexes to accommodate different size complexes. Significance StatementThe molecular mechanism by which Ycf1 transports a broad array of substrates that are essential for cellular detoxification and redox homeostasis remains unknown in the field of cellular biology. Here, guided by the novel substrate bound structure of Ycf1, we discovered a bipartite binding mechanism that accommodates substrates of varying sizes while maintaining specificity. Four crucial ionic interactions govern substrate specificity by recognizing ligands with a glutathione moiety, complemented by a sizable pocket on the adjacent side for different glutathione complexes.

biochemistry↗

bioRxiv2023

Structural Basis for Autoinhibition by the Dephosphorylated Regulatory Domain of Ycf1

Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected helix-strand hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.

biochemistry↗

bioRxiv2021

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

Yeast Cadmium Factor-1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4[A] and 4.0[A] in distinct inward-facing open conformations capturing a previously unobserved ordered state of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the lasso domain. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide long-sought after insights into how R-domains control ABCC transporter activity.

biochemistry↗

bioRxiv2021

Cryo-EM structure of an elusive pre-transport intermediate of the multidrug transporter BmrCD reveals modes of asymmetric drug binding

Vectorial substrate efflux by ATP binding cassette (ABC) transporters, which play a major role in multidrug resistance, entails the ATP-powered interconversion of the transporter between stable intermediates. Despite recent progress in structure elucidation of ABC transporters, a number of such intermediates have yet to be visualized and mechanistically interpreted. Here, we combine single particle cryo-EM, Double Electron Electron Resonance (DEER) spectroscopy with Molecular Dynamics simulations to profile and mechanistically frame the conformation of a hitherto unobserved intermediate in the context of BmrCD, a heterodimeric multidrug ABC exporter from Bacillus subtilis. In our cryo-EM structure, BmrCD adopts an inward-facing architecture bound to both ATP and the substrate Hoechst-33342 and is capped by an extracellular domain which undergoes ATP-dependent conformational changes. A striking feature of the structure is a symmetric arrangement of the nucleotide-binding domain (NBD) in the presence of ATP whereas binding of Hoechst at two distinct sites in an acidic pocket stabilizes an asymmetric arrangement of the transmembrane domain architecture (TMD). Mutation of residues coordinating Hoechst in the structure abrogates the cooperative stimulation of ATP hydrolysis. In conjunction with previous studies, our findings suggest a mechanistic role for symmetry mismatch between NBDs and TMDs in the conformational cycle of ABC transporters. Moreover, the resolved structures of bimodally-bound drugs are of notable importance for future rational design and optimization of molecules for targeted transport inhibition of ABC transporters. ONE SENTENCE SUMMARYCryo-EM and EPR analysis reveal cooperative substrate binding in BmrCD in an architecture primed for transport.

biophysics↗

bioRxiv2020

The peptide sensor motifs asymmetrically couple ATP hydrolysis to transport in the heterodimeric ABC transporter TmrAB

The ATP binding cassette (ABC) family of transporters move diverse small molecules across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The allosteric mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) presents an unresolved question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved motif called the peptide sensor in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the peptide sensor motif alter ATP hydrolysis rates as well as substrate release. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects likely destabilize a network between the peptide sensor motif and the Q-loop and X-loop, two known allosteric elements in the NBDs. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the peptide sensor motifs help coordinate the transition of TmrAB to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.

biochemistry↗