CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.

PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination.

Protein post-translation modification plays an important role in regulating DNA repair; however, the role of arginine methylation in this process is poorly understood. Here we identify the arginine methyltransferase PRMT5 as a key regulator of homologous recombination (HR)-mediated double-strand break (DSB) repair, which is mediated through its ability to methylate RUVBL1, a cofactor of the TIP60 complex. We show that PRMT5 targets RUVBL1 for methylation at position R205, which facilitates TIP60-dependent mobilization of 53BP1 from DNA breaks, promoting HR. Mechanistically, we demonstrate that PRMT5-directed methylation of RUVBL1 is critically required for the acetyltransferase activity of TIP60, promoting histone H4K16 acetylation, which facilities 53BP1 displacement from DSBs. Interestingly, RUVBL1 methylation did not affect the ability of TIP60 to facilitate ATM activation. Taken together, our findings reveal the importance of PRMT5-mediated arginine methylation during DSB repair pathway choice through its ability to regulate acetylation-dependent control of 53BP1 localization.

Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.

The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.

Identification of novel candidate predisposing genes in familial nonmedullary thyroid carcinoma implicating DNA damage repair pathways.

The genetic basis of nonsyndromic familial nonmedullary thyroid carcinoma (FNMTC) is still poorly understood, as the susceptibility genes identified so far only account for a small percentage of the genetic burden. Recently, germline mutations in DNA repair-related genes have been reported in cases with thyroid cancer. In order to clarify the genetic basis of FNMTC, 94 genes involved in hereditary cancer predisposition, including DNA repair genes, were analyzed in 48 probands from FNMTC families, through targeted next-generation sequencing (NGS). Genetic variants were selected upon bioinformatics analysis and in silico studies. Structural modeling and network analysis were also performed. In silico results of NGS data unveiled likely pathogenic germline variants in 15 families with FNMTC, in genes encoding proteins involved in DNA repair (ATM, CHEK2, ERCC2, BRCA2, ERCC4, FANCA, FANCD2, FANCF, and PALB2) and in the DICER1, FLCN, PTCH1, BUB1B, and RHBDF2 genes. Structural modeling predicted that most missense variants resulted in the disruption of networks of interactions between residues, with implications for local secondary and tertiary structure elements. Functional annotation and network analyses showed that the involved DNA repair proteins functionally interact with each other, within the same DNA repair pathway and across different pathways. MAPK activation was a common event in tumor progression. This study supports that rare germline variants in DNA repair genes may be accountable for FNMTC susceptibility, with potential future utility in patients' clinical management, and reinforces the relevance of DICER1 in disease etiology.

NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis.

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.

Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance.

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.

RuvbL1 and RuvbL2 enhance aggresome formation and disaggregate amyloid fibrils.

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.

Insights into the Structures of Superoxide Reductases from the Symbionts Ignicoccus hospitalis and Nanoarchaeum equitans.

Superoxide reductases (SORs) are enzymes that detoxify the superoxide anion through its reduction to hydrogen peroxide and exist in both prokaryotes and eukaryotes. The substrate is transformed at an iron catalytic center, pentacoordinated in the ferrous state by four histidines and one cysteine. SORs have a highly conserved motif, (E)(K)HxP-, in which the glutamate is associated with a redox-driven structural change, completing the octahedral coordination of the iron in the ferric state, whereas the lysine may be responsible for stabilization and donation of a proton to catalytic intermediates. We aimed to understand at the structural level the role of these two residues, by determining the X-ray structures of the SORs from the hyperthermophilic archaea Ignicoccus hospitalis and Nanoarchaeum equitans that lack the quasi-conserved lysine and glutamate, respectively, but have catalytic rate constants similar to those of the canonical enzymes, as we previously demonstrated. Furthermore, we have determined the crystal structure of the E23A mutant of I. hospitalis SOR, which mimics several enzymes that lack both residues. The structures revealed distinct structural arrangements of the catalytic center that simulate several catalytic cycle intermediates, namely, the reduced and the oxidized forms, and the glutamate-free and deprotonated ferric forms. Moreover, the structure of the I. hospitalis SOR provides evidence for the presence of an alternative lysine close to the iron center in the reduced state that may be a functional substitute for the "canonical" lysine.

Production and characterization of a novel Delta-like 1 functional unit as a tool for Notch pathway activation and generation of a specific antibody.

Notch signalling is an evolutionary conserved cell-to-cell communication pathway crucial for development and tissue homeostasis. Abnormal Notch signalling by mutations or deregulated expression of its receptors and/or ligands can lead to cancer making it a potential therapeutic target. Delta-like1 (DLL1) is a ligand of the Notch pathway implicated in different types of cancer, including breast cancer. Herein, we produced rhDLL1-DE3, a novel soluble form of DLL1 protein, which contains the DSL domain and EGF1-3 repeats critical for Notch pathway activation. cDNA fragments of human DLL1, encoding truncated versions of DLL1 with regions required to activate Notch receptors, were cloned and expressed as histidine-fused proteins in bacterial and mammalian cells. Expression tests in mammalian cells showed almost exclusively expression of the rhDLL1-DE3 protein form comprising the minimal binding regions DSL to EGF3 to Notch receptors. The highest yield of rhDLL1-DE3 was obtained from E. coli inclusion bodies. The produced protein, with purity higher than 95% bound to human Notch1 recombinant protein, by both Biolayer interferometry and ELISA assays. Cellular assays revealed rhDLL1-DE3 was biologically active as it increased expression of Notch-dependent genes in inducible pluripotent and breast cancer cells. Moreover, rhDLL1-DE3 allowed the generation of polyclonal antibodies by immunization that efficiently recognized DLL1 proteins by immunoblot, and caused a significant decrease of Notch1 expression in MCF7 breast cancer cells. The rhDLL1-DE3 protein might thus be used for Notch pathway activation and to generate anti-DLL1 monoclonal antibodies by immunization or phage display technology to unveil the effect of DLL1 in breast cancer.

Superoxide reductase from Giardia intestinalis: structural characterization of the first SOR from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction.

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage.

A unique glyceryl diglycoside identified in the thermophilic, radiation-resistant bacterium Rubrobacter xylanophilus.

The solute pool of the actinobacterium Rubrobacter xylanophilus has been investigated as a function of the growth temperature and concentration of NaCl in the medium (Empadinhas et al. Extremophiles 11: 667-673, 2007). Changing the carbon source from glucose to maltose in a minimal growth medium led to the accumulation of an unknown organic compound whose structure was investigated by NMR and confirmed by chemical synthesis in the present study as: (2R)-2-(1-O-α-D-mannopyranosyl)-3-(1-O-α-D-glucopyranosyl)-D-glycerate (MGlyG). In addition to this newly identified diglycoside, the solute pool of R. xylanophilus included trehalose, mannosylglycerate, di-myo-inositol phosphate and di-N-acetyl-glucosamine phosphate. The structure of MGlyG was established by NMR and confirmed by chemical synthesis. The availability of g-amounts of the synthetic material allowed us to perform stabilization tests on three model enzymes (malate dehydrogenase, staphylococcal nuclease, and lysozyme), and compare the efficacy of MGlyG with other natural glyceryl glycosides, such as α-D-mannosyl-D-glycerate, α-D-glucosyl-D-glycerate and α-D-glucosyl-(1 → 6)-α-D-glucosyl-(1 → 2)-D-glycerate.

Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions.

Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.

Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay.

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.

SAD phasing towards structure determination of a thermostable Rieske ferredoxin with a novel stabilizing disulfide bridge.

Rieske proteins and Rieske ferredoxins are ubiquitous electron-transfer metalloproteins that are characterized by a [2Fe-2S] cluster coordinated by pairs of cysteine and histidine residues. The thermoacidophilic archaeon Acidianus ambivalens contains a Rieske ferredoxin termed RFd2, which has an hitherto unknown additional region of 40-44 residues at the C-terminus with a Cx3C motif that introduces a novel disulfide bond within the Rieske fold. RFd2 was crystallized with the aim of determining its three-dimensional structure in order to understand the contribution of this as yet unique disulfide bridge to the function and stability of RFd2. RFd2 crystals were successively improved, increasing their diffraction to 1.9 Šresolution. Molecular replacement did not solve the RFd2 structure, but a highly multiple in-house diffraction data set collected at the Cu Kα edge led to solution of the phase problem.

Extract2Chip-Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance.

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.

Thermofluor-based optimization strategy for the stabilization and crystallization of Campylobacter jejuni desulforubrerythrin.

Desulforubrerythrin from Campylobacter jejuni has recently been biochemical and spectroscopically characterized. It is a member of the rubrerythrin family, and it is composed of three structural domains: the N-terminal desulforedoxin domain with a non-heme iron center, followed by a four-helix bundle domain harboring a binuclear iron center and finally a C-terminal rubredoxin domain. To date, this is the first example of a protein presenting this kind of structural domain organization, and therefore the determination of its crystal structure may unveil unexpected structural features. Several attempts were made in order to obtain protein crystals, but always without success. As part of our strategy the thermofluor method was used to increase protein stability and its propensity to crystallize. This approach has been recently used to optimize protein buffer formulation, thus yielding more stable and homogenous protein samples. Thermofluor has also been used to identify cofactors/ligands or small molecules that may help stabilize native protein states. A successful thermofluor approach was used to select a pH buffer condition that allowed the crystallization of Campylobacter jejuni desulforubrerythrin, by screening both buffer pH and salt concentration. A buffer formulation was obtained which increased the protein melting temperature by 7°C relatively to the initial purification buffer. Desulforubrerythrin was seen to be stabilized by lower pH and high salt concentration, and was dialyzed into the new selected buffer, 100mM MES pH 6.2, 500mM NaCl. This stability study was complemented with a second thermofluor assay in which different additives were screened. A crystallization screening was carried out and protein crystals were rapidly obtained in one condition. Protein crystal optimization was done using the same additive screening. Interestingly, a correlation between the stability studies and crystallization experiments using the additive screening could be established. The work presented here shows an elegant example where thermofluor was shown to be a key biophysical method that allowed the identification of an improved buffer formulation and the applicability of this technique to increase the propensity of a protein to crystallize is discussed.

Optimization of TEAD P-Site Binding Fragment Hit into In Vivo Active Lead MSC-4106.

The dysregulated Hippo pathway and, consequently, hyperactivity of the transcriptional YAP/TAZ-TEAD complexes is associated with diseases such as cancer. Prevention of YAP/TAZ-TEAD triggered gene transcription is an attractive strategy for therapeutic intervention. The deeply buried and conserved lipidation pocket (P-site) of the TEAD transcription factors is druggable. The discovery and optimization of a P-site binding fragment (1) are described. Utilizing structure-based design, enhancement in target potency was engineered into the hit, capitalizing on the established X-ray structure of TEAD1. The efforts culminated in the optimized in vivo tool MSC-4106, which exhibited desirable potency, mouse pharmacokinetic properties, and in vivo efficacy. In close correlation to compound exposure, the time- and dose-dependent downregulation of a proximal biomarker could be shown.

Deciphering cellular and molecular determinants of human DPCD protein in complex with RUVBL1/RUVBL2 AAA-ATPases.

DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built a 3D model of DPCD. In addition, we used a variety of orthogonal biophysical techniques including small-angle X-ray scattering, structural mass spectrometry and electron microscopy to assess the molecular determinants of DPCD interaction with R1R2. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.

Structural and functional insights into a dodecameric molecular machine - the RuvBL1/RuvBL2 complex.

RuvBL1 (RuvB-like 1) and its homolog RuvBL2 are evolutionarily highly conserved AAA(+) ATPases essential for many cellular activities. They play an important role in chromatin remodeling, transcriptional regulation and DNA damage repair. RuvBL1 and RuvBL2 are overexpressed in different types of cancer and interact with major oncogenic factors, such as ß-catenin and c-Myc regulating their function. We solved the first three-dimensional crystal structure of the human RuvBL complex with a truncated domain II and show that this complex is competent for helicase activity. The structure reveals a dodecamer consisting of two heterohexameric rings with alternating RuvBL1 and RuvBL2 monomers bound to ADP/ATP, that interact with each other via the retained part of domain II. The dodecameric quaternary structure of the R1ΔDII/R2ΔDII complex observed in the crystal structure was confirmed by small-angle X-ray scattering analysis. Interestingly, truncation of domain II led to a substantial increase in ATP consumption of RuvBL1, RuvBL2 and their complex. In addition, we present evidence that DNA unwinding of the human RuvBL proteins can be auto-inhibited by domain II, which is not present in the homologous bacterial helicase RuvB. Our data give new insights into the molecular arrangement of RuvBL1 and RuvBL2 and strongly suggest that in vivo activities of these highly interesting therapeutic drug targets are regulated by cofactors inducing conformational changes via domain II in order to modulate the enzyme complex into its active state.

Superoxide reductase from Nanoarchaeum equitans: expression, purification, crystallization and preliminary X-ray crystallographic analysis.

Superoxide reductases (SORs) are the most recent oxygen-detoxification system to be identified in anaerobic and microaerobic bacteria and archaea. SORs are metalloproteins that are characterized by their possession of a catalytic nonhaem iron centre in the ferrous form coordinated by four histidine ligands and one cysteine ligand. Ignicoccus hospitalis, a hyperthermophilic crenarchaeon, is the only organism known to date to serve as a host for Nanoarchaeum equitans, a nanosized hyperthermophilic archaeon isolated from a submarine hot vent which completely depends on the presence of and contact with I. hospitalis cells for growth to occur. Similarly to I. hospitalis, N. equitans has a neelaredoxin (a 1Fe-type SOR) that keeps toxic oxygen species under control, catalysing the one-electron reduction of superoxide to hydrogen peroxide. Blue crystals of recombinant N. equitans SOR in the oxidized form (12.7 kDa, 109 residues) were obtained using polyethylene glycol (PEG 2000 MME) as precipitant. These crystals diffracted to 1.9 Å resolution at 100 K and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 51.88, b = 82.01, c = 91.30 Å. Cell-content analysis suggested the presence of four monomers in the asymmetric unit. The Matthews coefficient (V(M)) was determined to be 1.9 Å(3) Da(-1), corresponding to an estimated solvent content of 36%. Self-rotation function and native Patterson calculations suggested a tetramer with 222 point-group symmetry, similar to other 1Fe-SORs. The three-dimensional structure will be determined by the molecular-replacement method.