Biology subjects

Stoll, G. A.

Publications and source records attributed to Stoll, G. A..

4 recordsLinked to original sources

bioRxiv2023

Crystal structure and biochemical activity of the macro domain from rubella virus p150

Rubella virus remains a global health threat. Rubella infections during pregnancy can cause serious congenital pathology and no antiviral treatments are available. Rubella virus encodes a nonstructural polyprotein with RNA polymerase, methyltransferase, and papain-like cysteine protease activities, along with a putative macro domain of unknown function. Macro domains bind ADP-ribose adducts, a post-translational modification that plays a key role in host-virus conflicts. Some macro domains can also remove the mono-ADP-ribose adduct or degrade poly-ADP-ribose chains. Here, we report high-resolution crystal structures of the macro domain from rubella virus nonstructural protein p150, with and without ADP-ribose bound. The overall fold is most similar to macroD-type macro domains from various nonviral species. The specific composition and structure of the residues that poised for catalysis or coordinate ADP-ribose in the rubella virus macro domain are most similar to those of macro domains from alphaviruses. Isothermal calorimetry and enzymatic assays show that the rubella virus macro domain binds ADP-ribose in solution and has mono-ADP-ribosylhydrolase (de-MARylation) activity. IMPORTANCEOur work demonstrates that, like alpha- and coronaviruses, rubiviruses encode a mono-ADP-ribosylhydrolase with a structurally conserved macro domain fold to counteract MARylation by PARPs in the host innate immune response. Our structural data will guide future efforts to develop novel antiviral therapeutics against rubella or infections with related viruses.

biochemistry↗

bioRxiv2022

Structure and functional mapping of the KRAB-KAP1 repressor complex

Transposable elements (TEs) are a genetic reservoir from which new genes and regulatory elements can emerge. Expression of TEs can be pathogenic, however, and is tightly regulated. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) recruit co-repressor KRAB-associated protein 1 (KAP1/TRIM28) to regulate many TEs but how KRAB-ZFPs and KAP1 interact remains unclear. Here, we report the crystal structure of the KAP1 tripartite motif (TRIM) in complex with the KRAB domain from a human KRAB-ZFP, ZNF93. Structure-guided mutations in the KAP1-KRAB binding interface abolished repressive activity in an epigenetic TE silencing assay. Deposition of H3K9me3 over thousands of loci is lost genome-wide in cells expressing a KAP1 variant with mutations that abolish KRAB binding. Our work identifies and functionally validates the KRAB-KAP1 molecular interface, which forms the nexus of a transcriptional control axis vital to vertebrates and underpins programmable gene repression by CRISPRi.

biochemistry↗

bioRxiv2021

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread

The herpes simplex virus (HSV)-1 protein pUL21 is essential for efficient virus replication and dissemination. While pUL21 has been shown to promote multiple steps of virus assembly and spread, the molecular basis of its function remained unclear. Here we identify that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). pUL21 directs the dephosphorylation of cellular and virus proteins, including components of the viral nuclear egress complex, and we define a conserved non-canonical linear motif in pUL21 that is essential for PP1 recruitment. In vitro evolution experiments reveal that pUL21 directly antagonises the activity of the virus-encoded kinase pUS3, with growth and spread of pUL21 PP1-binding mutant viruses being restored when pUS3 activity is disrupted. This study shows that virus-directed phosphatase activity is essential for efficient herpesvirus assembly and spread, highlighting the fine balance between kinase and phosphatase activity required for optimal virus replication.

microbiology↗

bioRxiv2018

Structure of the tripartite motif of KAP1/TRIM28 identifies molecular interfaces required for transcriptional silencing of retrotransposons

Transcription of transposable elements is tightly regulated to prevent damage to the genome. The family of KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, which controls the assembly of an epigenetic silencing complex including histone H3K9 methyltransferase SETDB1. The chromatin remodeling activities of this complex repress transcription of the targeted transposable element and any adjacent genes. Here, we use biophysical and structural approaches to show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevented higher-order oligomerization without a significant loss of retrotransposon silencing activity in a cell-based assay, indicating that, in contrast to other TRIM family members, self-assembly is not essential for the function of KAP1. The crystal structure of the KAP1 RBCC dimer identifies the KRAB domain binding site, in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 RBCC motif responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.

biochemistry↗