Biology subjects

Susac, L.

Publications and source records attributed to Susac, L..

4 recordsLinked to original sources

bioRxiv2025

Single-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC transporter

ATP-binding cassette (ABC) transporters are molecular machines involved in diverse physiological processes, including antigen processing by TAP, a key component of adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes, yet the precise mechanism linking ATP binding to substrate movement remains unclear. Here, we employ a single-molecule FRET sensor to visualize single translocation events by individual ABC transporters, overcoming the limitations of ensemble averaging. This approach reveals that substrate transport is driven by a conformational switch from the inward-to the outward-facing state. Using a slow-turnover TmrAB variant, we demonstrate that ATP binding alone, even in the absence of Mg2+, is sufficient to drive a single round of peptide translocation. Cryo-EM structures of wild-type and slow-turnover TmrAB show that ATP binding induces the outward-facing conformation even without Mg2+. In wild-type TmrAB, this conformational transition supports a single translocation event, whereas Mg2+-dependent ATP hydrolysis is required to reset the transporter. These findings establish a direct mechanistic link between ATP binding and substrate translocation at single-molecule resolution, providing new insights into the catalytic cycle of ABC transporters.

biochemistry↗

bioRxiv2025

US6 hijacks the peptide-loading complex by trapping transporter-chaperone dynamics

Adaptive immune responses are initiated by major histocompatibility class I (MHC I) presentation of antigenic peptides on the cell surface. This process relies on the peptide-loading complex (PLC), a dynamic transporter-multichaperone assembly in the endoplasmic reticulum (ER), to ensure high-fidelity selection, editing, and loading of peptides onto MHC I heterodimers1. The PLC is the primary target for viral immune evasion2, elicited in particular by human cytomegalovirus (HCMV)3, causing lifelong infections with severe risks for immunocompromised individuals. While the overall architecture of the PLC is known4, how its activity is jeopardized by viral immune evasins remains unclear. Here, we present the 2.59-2.88 [A] cryogenic electron microscopy structure of native human PLC associated with the HCMV immune evasin US6. US6 inhibits the heterodimeric transporter associated with antigen processing (TAP1/2) by latching its transmembrane helix laterally onto TAP2 and using its central disulfide-rich domain to mimic a translocating peptide. This effectively plugs the ER-lumenal exit and locks TAP in an outward-facing open conformation with closed nucleotide-binding domains and asymmetrically occluded ATP and ADP. The structure also highlights the role of the unique N-terminal transmembrane domains of TAP as dynamic scaffolds that recruit the MHC I-specific chaperone tapasin by clamping its transmembrane helix to the core transmembrane domain of each transporter subunit. Our findings uncover the molecular mechanism of US6-mediated viral immune evasion and reveal potential targets for therapeutic modulation of antigen presentation in cancer and infectious diseases.

immunology↗

bioRxiv2022

Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN

The heterotetrameric human transfer RNA (tRNA) splicing endonuclease (TSEN) catalyzes the excision of intronic sequences from precursor tRNAs (pre-tRNAs)1. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH)2-8. The three-dimensional (3D) assembly of TSEN/CLP1, the mechanism of substrate recognition, and the molecular details of PCH-associated mutations are not fully understood. Here, we present cryo-electron microscopy structures of human TSEN with intron-containing pre-tRNATyrO_SCPLOWGTAC_SCPLOW and pre-tRNAArgO_SCPLOWTCTC_SCPLOW. TSEN exhibits broad structural homology to archaeal endonucleases9 but has evolved additional regulatory elements that are involved in handling and positioning substrate RNA. Essential catalytic residues of subunit TSEN34 are organized for the 3 splice site which emerges from a bulge-helix configuration. The triple-nucleotide bulge at the intron/3-exon boundary is stabilized by an arginine tweezer motif of TSEN2 and an interaction with the proximal minor groove of the helix. TSEN34 and TSEN54 define the 3 splice site by holding the tRNA body in place. TSEN54 adapts a bipartite fold with a flexible central region required for CLP1 binding. PCH-associated mutations are located far from pre-tRNA binding interfaces explaining their negative impact on structural integrity of TSEN without abrogating its catalytic activity in vitro10. Our work defines the molecular framework of pre-tRNA recognition and cleavage by TSEN and provides a structural basis to better understand PCH in the future.

biochemistry↗

bioRxiv2021

Structure of telomerase-bound CST with Polymerase α-Primase

Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3-overhang of the G-strand and a group of proteins called shelterin1,2. Among these, TPP1 and POT1 associate with the 3-overhang, with POT1 binding the G-strand3 and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT)4. The ends of the telomeric DNA are replicated and maintained by telomerase5, for the G-strand, and subsequently DNA Polymerase -Primase6,7 (PolPrim), for the C-strand8. PolPrim is stimulated by CTC1-STN1-TEN1 (CST)9-12, but the structural basis of both PolPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolPrim, as well as of PolPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex--telomerase core RNP, p50/TPP1, CST and PolPrim--that provides unprecedented insights into CST and PolPrim recruitment and handoff between G-strand and C-strand synthesis.

molecular biology↗