Structure of STAU1 dsRBD4 and dsRBD3/4 in complex with sARF1 SBS dsRNA. (A) Structural ensemble of the STAU1 dsRBD4–sARF1 SBS dsRNA complex. Heavy-atom superposition of the ensemble of the 20 lowest-energy structures. The protein backbone is shown in dark green and the RNA heavy atoms of the bases in orange and those of the ribose-phosphodiester backbone are shown in gold (omitting phosphate and 2’-OH oxygens). (B) Schematic representation of sARF1 SBS dsRNA showing interactions of dsRBD3 as well as dsRBD4 as dotted lines. Interactions with the ribose-phosphodiester backbone are circled in dark green for dsRBD4 and light green for dsRBD3 while base interactions are shown as filled circles.

Peter J. Lukavsky Research Group

Significance

Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3’ UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. The structure of the ARF1 SBS–STAU1 complex, presented in this study, uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix a1, the b1–b 2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix a2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitrobinding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.

Yadav, DP, Zigáčková, D., Zlobina, M.,  Klumpler, Beaumont, TC., Kubíčková, M., Vaňáčová, Š,  and Lukavsky, PJ.: Staufen1 reads out structure and sequence features in ARF1 dsRNA for target recognition,  Nucleic Acids Res.  2020, 48, 2091-2106, doi:10.1093/nar/gkz1163

Crystal structures of (A) PBD anthramycin covalently bound to DNA strands and (B) lincomycin targeting the peptidyl transferase center in the 50S ribosomal subunit of Staphylococcus aureus.

Jiří Janata Research Group

Significance

Antitumor pyrrolobenzodiazepines (PBDs), lincosamide antibiotics, quorumsensing molecule hormaomycin, and antimicrobial griselimycin are structurally and functionally diverse groups of actinobacterial metabolites. The common feature of these compounds is the incorporation of L -tyrosine- or L -leucine-derived 4-alkyl-L -proline derivatives (APDs) in their structures. In this study, the authors report that the last reaction in the biosynthetic pathway of APDs, catalyzed by F420 H2 -dependent Apd6 reductases, contributes to the structural diversity of APD precursors. Specifically, the heterologous overproduction of six Apd6 enzymes demonstrated that Apd6 from the biosynthesis of PBDs and hormaomycin can reduce only an endocyclic imine double bond, whereas Apd6 LmbY and partially GriH from the biosynthesis of lincomycin and griselimycin, respectively, also reduce the more inert exocyclic double bond of the same 4- substituted Δ 1-pyrroline-2-carboxylic acid substrate, making LmbY and GriH unusual, if not unique, among reductases. Furthermore, the differences in the reaction specificity of the Apd6 reductases determine the formation of the fully saturated APD moiety of lincomycin versus the unsaturated APD moiety of PBDs, providing molecules with optimal shapes to bind their distinct biological targets. Moreover, the Apd6 reductases establish the first F420 H2-dependent enzymes from the luciferase-like hydride transferase protein superfamily in the biosynthesis of bioactive molecules. Finally, bioinformatics analysis demonstrates that Apd6 and their homologues, widely distributed within several bacterial phyla, play a role in the formation of novel yet unknown natural products with incorporated L-proline-like precursors and likely in the microbial central metabolism.

Steiningerova, L. Kamenik, Z.*,  Gazak, R.,  Kadlcik, S.,  Bashiri, G.,  Man, P., Kuzma, M., Pavlikova, M., and Janata, J.: Different Reaction Specificities of F420H2‑Dependent Reductases Facilitate Pyrrolobenzodiazepines and Lincomycin To Fit Their Biological Targets, J. Am. Chem. Soc.  2020, 142, 3440-3448, https://dx.doi.org/10.1021/jacs.9b11234