Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes.

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

Structural basis of the key residue W320 responsible for Hsp90 conformational change.

The 90-kDa heat shock protein (Hsp90) is a homodimeric molecular chaperone with ATPase activity, which has become an intensely studied target for the development of drugs for the treatment of cancer, neurodegenerative and infectious diseases. The equilibrium between Hsp90 dimers and oligomers is important for modulating its function. In the absence of ATP, the passive chaperone activity of Hsp90 dimers and oligomers has been shown to stabilize client proteins as a holdase, which enhances substrate binding and prevents irreversible aggregation and precipitation of the substrate proteins. In the presence of ATP and its associated cochaperones, Hsp90 homodimers act as foldases with the binding and hydrolysis of ATP driving conformational changes that mediate client folding. Crystal structures of both wild type and W320A mutant Hsp90αMC (middle/C-terminal domain) have been determined, which displayed a preference for hexameric and dimeric states, respectively. Structural analysis showed that W320 is a key residue for Hsp90 oligomerization by forming intermolecular interactions at the Hsp90 hexameric interface through cation-π interactions with R367. W320A substitution results in the formation of a more open conformation of Hsp90, which has not previously been reported, and the induction of a conformational change in the catalytic loop. The structures provide new insights into the mechanism by which W320 functions as a key switch for conformational changes in Hsp90 self-oligomerization, and binding cochaperones and client proteins.Communicated by Ramaswamy H. Sarma.

Structure-Activity Relationship Study of Tertiary Alcohol Hsp90α-Selective Inhibitors with Novel Binding Mode.

The heat shock protein 90 (Hsp90) family of molecular chaperones mediates the folding and activation of client proteins associated with all 10 hallmarks of cancer. Herein, the design, synthesis, and biological validation of Hsp90α-selective inhibitors that contain a tertiary alcohol are reported. Forty-one analogues were synthesized to modulate hydrogen-bonding interactions and to probe for steric and hydrophobic interactions within the Hsp90α binding site. Cocrystal structures of lead compound 23d (IC50 = 0.25 µM, 15-fold selective vs Hsp90ß) and a 5-fluoroisoindoline derivative (KUNA-111) revealed a novel binding mode that induced conformational changes within Hsp90α's N-terminal domain. The lead Hsp90α-selective inhibitors did not manifest significant antiproliferative activity, but they did result in selective and dose-dependent degradation of Hsp90α clients in the cellular environment. Additional studies will be sought to determine the effects of the novel conformational change induced by 23d.

Crystal structure of the middle and C-terminal domains of Hsp90α labeled with a coumarin derivative reveals a potential allosteric binding site as a drug target.

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.

EF-hand protein, EfhP, specifically binds Ca2+ and mediates Ca2+ regulation of virulence in a human pathogen Pseudomonas aeruginosa.

Calcium (Ca2+) is well known as a second messenger in eukaryotes, where Ca2+ signaling controls life-sustaining cellular processes. Although bacteria produce the components required for Ca2+ signaling, little is known about the mechanisms of bacterial Ca2+ signaling. Previously, we have identified a putative Ca2+-binding protein EfhP (PA4107) with two canonical EF-hand motifs and reported that EfhP mediates Ca2+ regulation of virulence factors production and infectivity in Pseudomonas aeruginosa, a human pathogen causing life-threatening infections. Here, we show that EfhP selectively binds Ca2+ with 13.7 µM affinity, and that mutations at the +X and -Z positions within each or both EF-hand motifs abolished Ca2+ binding. We also show that the hydrophobicity of EfhP increased in a Ca2+-dependent manner, however no such response was detected in the mutated proteins. 15 N-NMR showed Ca2+-dependent chemical shifts in EfhP confirming Ca2+-binding triggered structural rearrangements in the protein. Deletion of efhP impaired P. aeruginosa survival in macrophages and virulence in vivo. Disabling EfhP Ca2+ binding abolished Ca2+ induction of pyocyanin production in vitro. These data confirm that EfhP selectively binds Ca2+, which triggers its structural changes required for the Ca2+ regulation of P. aeruginosa virulence, thus establishing the role of EfhP as a Ca2+ sensor.

Structure and function of an effector domain in antiviral factors and tumor suppressors SAMD9 and SAMD9L.

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 multidomain 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.

Structure of the unique tetrameric STENOFOLIA homeodomain bound with target promoter DNA.

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 Šresolution. 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.

Selective Inhibition of the Hsp90α Isoform.

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone that processes nascent polypeptides into their biologically active conformations. Many of these proteins contribute to the progression of cancer, and consequently, inhibition of the Hsp90 protein folding machinery represents an innovative approach toward cancer chemotherapy. However, clinical trials with Hsp90 N-terminal inhibitors have encountered deleterious side effects and toxicities, which appear to result from the pan-inhibition of all four Hsp90 isoforms. Therefore, the development of isoform-selective Hsp90 inhibitors is sought to delineate the pathological role played by each isoform. Herein, we describe a structure-based approach that was used to design the first Hsp90α-selective inhibitors, which exhibit >50-fold selectivity versus other Hsp90 isoforms.

A semi-dominant NLR allele causes whole-seedling necrosis in wheat.

Programmed cell death (PCD) and apoptosis have key functions in development and disease resistance in diverse organisms; however, the induction of necrosis remains poorly understood. Here, we identified a semi-dominant mutant allele that causes the necrotic death of the entire seedling (DES) of wheat (Triticum aestivum L.) in the absence of any pathogen or external stimulus. Positional cloning of the lethal allele mDES1 revealed that this premature death via necrosis was caused by a point mutation from Asp to Asn at amino acid 441 in a nucleotide-binding leucine-rich repeat protein containing nucleotide-binding domain and leucine-rich repeats. The overexpression of mDES1 triggered necrosis and PCD in transgenic plants. However, transgenic wheat harboring truncated wild-type DES1 proteins produced through gene editing that exhibited no significant developmental defects. The point mutation in mDES1 did not cause changes in this protein in the oligomeric state, but mDES1 failed to interact with replication protein A leading to abnormal mitotic cell division. DES1 is an ortholog of Sr35, which recognizes a Puccinia graminis f. sp. tritici stem rust disease effector in wheat, but mDES1 gained function as a direct inducer of plant death. These findings shed light on the intersection of necrosis, apoptosis, and autoimmunity in plants.

Identification of factors associated with duplicate rate in ChIP-seq data.

Chromatin immunoprecipitation and sequencing (ChIP-seq) has been widely used to map DNA-binding proteins, histone proteins and their modifications. ChIP-seq data contains redundant reads termed duplicates, referring to those mapping to the same genomic location and strand. There are two main sources of duplicates: polymerase chain reaction (PCR) duplicates and natural duplicates. Unlike natural duplicates that represent true signals from sequencing of independent DNA templates, PCR duplicates are artifacts originating from sequencing of identical copies amplified from the same DNA template. In analysis, duplicates are removed from peak calling and signal quantification. Nevertheless, a significant portion of the duplicates is believed to represent true signals. Obviously, removing all duplicates will underestimate the signal level in peaks and impact the identification of signal changes across samples. Therefore, an in-depth evaluation of the impact from duplicate removal is needed. Using eight public ChIP-seq datasets from three narrow-peak and two broad-peak marks, we tried to understand the distribution of duplicates in the genome, the extent by which duplicate removal impacts peak calling and signal estimation, and the factors associated with duplicate level in peaks. The three PCR-free histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq data had about 40% duplicates and 97% of them were within peaks. For the other datasets generated with PCR amplification of ChIP DNA, as expected, the narrow-peak marks have a much higher proportion of duplicates than the broad-peak marks. We found that duplicates are enriched in peaks and largely represent true signals, more conspicuous in those with high confidence. Furthermore, duplicate level in peaks is strongly correlated with the target enrichment level estimated using nonredundant reads, which provides the basis to properly allocate duplicates between noise and signal. Our analysis supports the feasibility of retaining the portion of signal duplicates into downstream analysis, thus alleviating the limitation of complete deduplication.

Structure of a lipid-bound viral membrane assembly protein reveals a modality for enclosing the lipid bilayer.

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.

Structure-guided design of an Hsp90ß N-terminal isoform-selective inhibitor.

The 90 kDa heat shock protein (Hsp90) is a molecular chaperone responsible for folding proteins that are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors of Hsp90 have entered clinical trials, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms. pan-Inhibition of Hsp90 appears to be detrimental as toxicities have been reported alongside induction of the pro-survival heat shock response. The development of Hsp90 isoform-selective inhibitors represents an alternative approach towards the treatment of cancer that may limit some of the detriments. Described herein is a structure-based approach to design isoform-selective inhibitors of Hsp90ß, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90ß-selective inhibitors as a method to overcome the detriments associated with pan-inhibition.

High-resolution crystal structure reveals a HEPN domain at the C-terminal region of S. cerevisiae RNA endonuclease Swt1.

Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomyces cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.

Crystal structure of uroporphyrinogen III synthase from Pseudomonas syringae pv. tomato DC3000.

Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole compounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III (uro'gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5Å resolution by the single wavelength anomalous dispersion (SAD) method. Each psU3S molecule consists of two domains interlinked by a two-stranded antiparallel ß-sheet. The conformation of psU3S is different from its homologous proteins because of the flexibility of the linker between the two domains, which might be related to this enzyme's catalytic properties. Based on mutation and activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S.