Biology subjects

Kroupova, A.

Publications and source records attributed to Kroupova, A..

5 recordsLinked to original sources

bioRxiv2025

Structural framework for the assembly of the human tRNA ligase complex

In human cells, a subset of tRNA-encoding genes contain introns. These are removed by a non-canonical splicing pathway in which the tRNA splicing endonuclease complex catalyzes intron excision and the resulting exons are subsequently ligated by the tRNA-ligase complex (tRNA-LC). Although recent studies have provided insights into the process of intron removal, the molecular mechanisms underpinning tRNA ligation by tRNA-LC remain elusive. The tRNA-LC is a hetero-pentameric protein assembly consisting of Ashwin, CGI-99, FAM98B, the DEAD-box helicase DDX1 and the catalytic subunit RTCB/HSPC117. Using cryo-EM, we have determined an atomic-resolution reconstruction of human tRNA-LC. We find that CGI-99, DDX1 and FAM98B form an alpha-helical bundle that contacts RTCB via an interface located on the opposite side from the location of the ligase active site and tethers DDX1 to the tRNA-LC via its C-terminal helix. FAM98B and CGI-99 extensively interact in an intricately co-folded heterodimer that clamps Ashwin in a pincer-like structure. Interaction analysis using structure-based mutants of tRNA-LC subunits supports the overall architecture of the complex. Finally, we show that the paralogous proteins FAM98A and FAM98C underpin the assembly of compositionally distinct RTCB-containing complexes that lack Ashwin and may have distinct cellular functions. Together, our results provide new insights into the assembly and mechanism of the tRNA ligase complex, shedding light on its functions in tRNA biogenesis and beyond.

biochemistry↗

bioRxiv2025

A method for the detection and enrichment of endogenous cereblon substrates

C-Terminal cyclic imides are posttranslational modifications on proteins that are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN). Despite the observation of these modifications across the proteome by mass spectrometry-based proteomics, an orthogonal and generalizable method to visualize the C-terminal cyclic imide would enhance detection, sensitivity, and throughput of endogenous CRBN substrate characterization. Here we develop an antibody-like reagent, termed "cerebody," for visualizing and enriching C-terminal cyclic imide-modified proteins. We describe the engineering of CRBN derivatives to produce cerebody and use it to identify CRBN substrates by Western blot and enrichment from whole cell and tissue lysates. CRBN substrates identified by cerebody enrichment are mapped, validated, and further characterized for dependence on the C-terminal cyclic imide modification. These methods will accelerate the characterization of endogenous CRBN substrates and their regulation.

biochemistry↗

bioRxiv2024

Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degraders ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics. One sentence summaryA novel Cereblon construct (CRBNmidi) allows structural and biophysical enablement of ligand and degrader design

biochemistry↗

bioRxiv2023

Mechanistic basis for oxidative stress protection of the human tRNA ligase complex by the oxidoreductase PYROXD1

RTCB is the catalytic subunit of the metazoan tRNA ligase complex (tRNA-LC) that plays essential roles in tRNA biogenesis and unfolded protein response. The catalytic center of RTCB contains a conserved cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of mammalian tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here we report a cryo-EM structure of human RTCB in complex with PYROXD1, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its C-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment and PYROXD1, while PYROXD1 reoxidation enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of tRNA-LC under aerobic conditions.

biochemistry↗

bioRxiv2021

Molecular architecture of the human tRNA ligase complex

RtcB enzymes are RNA ligases that play essential roles in tRNA splicing, unfolded protein response, and RNA repair. In metazoa, RtcB functions as part of a five-subunit tRNA ligase complex (tRNA-LC) along with Ddx1, Cgi-99, Fam98B and Ashwin. The human tRNA-LC or its individual subunits have been implicated in additional cellular processes including microRNA maturation, viral replication, DNA double-strand break repair and mRNA transport. Here we present a biochemical analysis of the inter-subunit interactions within the human tRNA-LC along with crystal structures of the catalytic subunit RTCB and the N-terminal domain of CGI-99. We show that the core of the human tRNA-LC is assembled from RTCB and the C-terminal alpha-helical regions of DDX1, CGI-99, and FAM98B, all of which are required for complex integrity. The N-terminal domain of CGI-99 displays structural homology to calponin-homology domains, and CGI-99 and FAM98B associate via their N-terminal domains to form a stable subcomplex. The crystal structure of GMP-bound RTCB reveals divalent metal coordination geometry in the active site, providing insights into its catalytic mechanism. Collectively, these findings shed light on the molecular architecture and mechanism of the human tRNA ligase complex, and provide a structural framework for understanding its functions in cellular RNA metabolism.

biochemistry↗