SAMD9 and SAMD9L (SAMD9/9L) are antiviral factors and tumor suppressors, playing a critical role in innate immune defense against poxviruses and the development of myeloid tumors. SAMD9/9L mutations with a gain-of-function (GoF) in inhibiting cell growth cause multisystem developmental disorders including many pediatric myelodysplastic syndromes. Predicted to be multi-domain proteins with an architecture like that of the NOD-like receptors, SAMD9/9L molecular functions and domain structures are largely unknown. Here, we identified a SAMD9/9L effector domain that functions by binding to double-stranded nucleic acids (dsNA) and determined the crystal structure of the domain in complex with DNA. Aided with precise mutations that differentially perturb dsNA binding, we demonstrated that the antiviral and antiproliferative functions of the wild-type and GoF SAMD9/9L variants rely on dsNA binding by the effector domain. Furthermore, we showed that GoF variants inhibit global protein synthesis, reduce translation elongation, and induce proteotoxic stress response, which all require dsNA binding by the effector domain. The identification of the structure and function of a SAMD9/9L effector domain provides a therapeutic target for SAMD9/9L-associated human diseases.

The 90 kDa heat-shock protein (Hsp90) is an abundant molecular chaperone that is essential to activate, stabilize and regulate the function of a plethora of client proteins. As drug targets for the treatment of cancer and neurodegenerative diseases, Hsp90 inhibitors that bind to the N-terminal ATP-binding site of Hsp90 have shown disappointing efficacy in clinical trials. Thus, allosteric regulation of the function of Hsp90 by compounds that interact with its middle and C-terminal (MC) domains is now being pursued as a mechanism to inhibit the ATPase activity and client protein-binding activity of Hsp90 without concomitant induction of the heat-shock response. Here, the crystal structure of the Hsp90αMC protein covalently linked to a coumarin derivative, MDCC {7-diethylamino-3-[N-(2-maleimidoethyl)carbamoyl]coumarin}, which is located in a hydrophobic pocket that is formed at the Hsp90αMC hexamer interface, is reported. MDCC binding leads to the hexamerization of Hsp90, and the stabilization and conformational changes of three loops that are critical for its function. A fluorescence competition assay demonstrated that other characterized coumarin and isoflavone-containing Hsp90 inhibitors compete with MDCC binding, suggesting that they could bind at a common site or that they might allosterically alter the structure of the MDCC binding site. This study provides insights into the mechanism by which the coumarin class of allosteric inhibitors potentially disrupt the function of Hsp90 by regulating its oligomerization and the burial of interaction sites involved in the ATP-dependent folding of Hsp90 clients. The hydrophobic binding pocket characterized here will provide new structural information for future drug design.

Homeobox transcription factors are key regulators of morphogenesis and development in both animals and plants. In plants, the WUSCHEL-related homeobox (WOX) family of transcription factors function as central organizers of several developmental programs ranging from embryo patterning to meristematic stem-cell maintenance through transcriptional activation and repression mechanisms. The Medicago truncatula STENOFOLIA (STF) gene is a master regulator of leaf-blade lateral development. Here, the crystal structure of the homeodomain (HD) of STF (STF-HD) in complex with its promoter DNA is reported at 2.1 Å resolution. STF-HD binds DNA as a tetramer, enclosing nearly the entire bound DNA surface. The STF-HD tetramer is partially stabilized by docking of the C-terminal tail of one protomer onto a conserved hydrophobic surface on the head of another protomer in a head-to-tail manner. STF-HD specifically binds TGA motifs, although the promoter sequence also contains TAAT motifs. Helix α3 not only serves a canonical role as a base reader in the major groove, but also provides DNA binding in the minor groove through basic residues located at its C-terminus. The structural and functional data in planta reported here provide new insights into the DNA-binding mechanisms of plant-specific HDs from the WOX family of transcription factors.

Leaf is a primary organ for the plants to sustain their life and support life on earth by assimilating solar energy and carbon dioxide into chemical energy. STENOFOLIA (STF), a WUSCHEL (WUS)‐like homeobox transcriptional regulator, plays a key role in embryonic patterning, blade outgrowth, and leaf vascular patterning. STF has also been shown to help in increasing biomass and solubilized sugar yield. STF does not affect leaf outgrowth directly but is critical for their development. It is known that transcriptional repressor activity mediated by the WUSbox of STF acts to promote blade outgrowth, and STF executes its functions with its interactions with other protein and DNA. STF binds to GA rich DNA sequence and there is specific DNA binding domain present in the sequence. Although the DNA binding domain (HD) of the protein is conserved across the species, the WOX family HD seems to be unique and cannot be replaced by another HD protein from the plant. WOX HD domain is longer than usual HD domains and has additional protein interaction sites to achieve stable repressive activity. The molecular mechanism of STF function and how WOX genes have adopted the WUS in evolutionary development are unknown. Here we are working towards determining the atomic structure of STF in complex with DNA by using x‐ray crystallography.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular membranes are maintained as closed compartments, broken up only transiently during membrane reorganization or lipid transportation. However, open-ended membranes, likely derived from scissions of the endoplasmic reticulum, persist in vaccinia virus-infected cells during the assembly of the viral envelope. A group of viral membrane assembly proteins (VMAPs) were identified as essential for this process. To understand the mechanism of VMAPs, we determined the 2.2-Å crystal structure of the largest member, named A6, which is a soluble protein with two distinct domains. The structure of A6 displays a novel protein fold composed mainly of alpha helices. The larger C-terminal domain forms a unique cage that encloses multiple glycerophospholipids with a lipid bilayer-like configuration. The smaller N-terminal domain does not bind lipid but negatively affects lipid binding by A6. Mutations of key hydrophobic residues lining the lipid-binding cage disrupt lipid binding and abolish viral replication. Our results reveal a protein modality for enclosing the lipid bilayer and provide molecular insight into a viral machinery involved in generating and/or stabilizing open-ended membranes.