Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action.

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Long-term testosterone therapy improves liver parameters and steatosis in hypogonadal men: a prospective controlled registry study.

Non-alcoholic fatty liver disease (NAFLD) is associated with cardiovascular disease (CVD) and both are prevalent in men with testosterone deficiency. Long-term effects of testosterone therapy (TTh) on NAFLD are not well studied. This observational, prospective, cumulative registry study assesses long-term effects of testosterone undecanoate (TU) on hepatic physiology and function in 505 hypogonadal men (T levels ≤350 ng/dL). Three hundred and twenty one men received TU 1000 mg/12 weeks for up to 12 years following an initial 6-week interval (T-group), while 184 who opted against TTh served as controls (C-group). T-group patients exhibited decreased fatty liver index (FLI, calculated according to Mayo Clinic guidelines) (83.6 ± 12.08 to 66.91 ± 19.38), γ-GT (39.31 ± 11.62 to 28.95 ± 7.57 U/L), bilirubin (1.64 ± 4.13 to 1.21 ± 1.89 mg/dL) and triglycerides (252.35 ± 90.99 to 213 ± 65.91 mg/dL) over 12 years. Waist circumference and body mass index were also reduced in the T-group (107.17 ± 9.64 to 100.34 ± 9.03 cm and 31.51 ± 4.32 to 29.03 ± 3.77 kg/m2). There were 25 deaths (7.8%) in the T-group of which 11 (44%) were cardiovascular related. In contrast, 28 patients (15.2%) died in C-group, and all deaths (100%) were attributed to CVD. These data suggest that long-term TTh improves hepatic steatosis and liver function in hypogonadal men. Improvements in liver function may have contributed to reduced CVD-related mortality.

Structure of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium vivax sheds light on drug resistance.

The genomes of the malaria-causing Plasmodium parasites encode a protein fused of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) domains that catalyze sequential reactions in the folate biosynthetic pathway. Whereas higher organisms derive folate from their diet and lack the enzymes for its synthesis, most eubacteria and a number of lower eukaryotes including malaria parasites synthesize tetrahydrofolate via DHPS. Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) HPPK-DHPSs are currently targets of drugs like sulfadoxine (SDX). The SDX effectiveness as an antimalarial drug is increasingly diminished by the rise and spread of drug-resistant mutations. Here, we present the crystal structure of PvHPPK-DHPS in complex with four substrates/analogs, revealing the bifunctional PvHPPK-DHPS architecture in an unprecedented state of enzymatic activation. SDX's effect on HPPK-DHPS is due to 4-amino benzoic acid (pABA) mimicry, and the PvHPPK-DHPS structure sheds light on the SDX-binding cavity, as well as on mutations that effect SDX potency. We mapped five dominant drug resistance mutations in PvHPPK-DHPS: S382A, A383G, K512E/D, A553G, and V585A, most of which occur individually or in clusters proximal to the pABA-binding site. We found that these resistance mutations subtly alter the intricate enzyme/pABA/SDX interactions such that DHPS affinity for pABA is diminished only moderately, but its affinity for SDX is changed substantially. In conclusion, the PvHPPK-DHPS structure rationalizes and unravels the structural bases for SDX resistance mutations and highlights architectural features in HPPK-DHPSs from malaria parasites that can form the basis for developing next-generation anti-folate agents to combat malaria parasites.

Optimization of Zika virus envelope protein production for ELISA and correlation of antibody titers with virus neutralization in Mexican patients from an arbovirus endemic region.

BACKGROUND: Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications. METHODS: We designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera. RESULTS: Replacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro. CONCLUSIONS: Results obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.

Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei.

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.

Effects of testosterone replacement therapy withdrawal and re-treatment in hypogonadal elderly men upon obesity, voiding function and prostate safety parameters.

Whether testosterone replacement therapy (TRT) is a lifelong treatment for men with hypogonadism remains unknown. We investigated long-term TRT and TRT withdrawal on obesity and prostate-related parameters. Two hundred and sixty-two hypogonadal patients (mean age 59.5) received testosterone undecanoate in 12-week intervals for a maximum of 11 years. One hundred and forty-seven men had TRT interrupted for a mean of 16.9 months and resumed thereafter (Group A). The remaining 115 patients were treated continuously (Group B). Prostate volume, prostate-specific antigen (PSA), residual voiding volume, bladder wall thickness, C-reactive protein (CRP), aging male symptoms (AMS), International Index of erectile function - erectile function (IIEF-EF) and International Prostate Symptoms Scores (IPSS) were measured over the study period with anthropometric parameters of obesity, including weight, body mass index (BMI) and waist circumference. Prior to interruption, TRT resulted in improvements in residual voiding volume, bladder wall thickness, CRP, AMS, IIEF-EF, IPSS and obesity parameters while PSA and prostate volume increased. TRT interruption reduced total testosterone to hypogonadal levels in Group A and resulted in worsening of obesity parameters, AMS, IPSS, residual voiding volume and bladder wall thickness, IIEF-EF and PSA while CRP and prostate volume were unchanged until treatment resumed whereby these effects were reversed. TRT interruption results in worsening of symptoms. Hypogonadism may require lifelong TRT.

Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo(5)U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo(5)U and was annotated as an S-adenosyl-L-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-L-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry 1im8, suggests that the active site contains SCM-SAH and not SAM.

A streamlined implementation of the glutamine synthetase-based protein expression system.

BACKGROUND: The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting. RESULTS: Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an "average" clone and ~40% that of the "best" clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein. CONCLUSION: Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where <10 mg/L of protein expression is often required but relatively large numbers of constructs frequently need to be trialed.

Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment.

BACKGROUND: Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS: We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPß. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPß. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 µM). CONCLUSION: The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Outrunning free radicals in room-temperature macromolecular crystallography.

A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A(2A) adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.

In situ macromolecular crystallography using microbeams.

Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Šresolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and ß D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Structural mass spectrometry decodes domain interaction and dynamics of the full-length Human Histone Deacetylase 2.

Human Histone Deacetylase 2 (HDAC2) belongs to a conserved enzyme superfamily that regulates deacetylation inside cells. HDAC2 is a drug target as it is known to be upregulated in cancers and neurodegenerative disorders. It consists of globular deacetylase and C-terminus intrinsically-disordered domains [1-3]. To date, there is no full-length structure of HDAC2 available due to the high intrinsic flexibility of its C-terminal domain. The intrinsically-disordered domain, however, is known to be important for the enzymatic function of HDAC2 [1, 4]. Here we combine several structural Mass Spectrometry (MS) methodologies such as denaturing, native, ion mobility and chemical crosslinking, alongside biochemical assays and molecular modelling to study the structure and dynamics of the full-length HDAC2 for the first time. We show that MS can easily dissect heterogeneity inherent within the protein sample and at the same time probe the structural arrangement of the different conformers present. Activity assays combined with data from MS and molecular modelling suggest how the structural dynamics of the C-terminal domain, and its interactions with the catalytic domain, regulate the activity of this enzyme.

The structure of nontypeable Haemophilus influenzae SapA in a closed conformation reveals a constricted ligand-binding cavity and a novel RNA binding motif.

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.

Temperature-dependent secretion of Zika virus envelope and non-structural protein 1 in mammalian cells for clinical applications.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus associated with congenital Zika syndrome and Guillain-Barré syndrome in adults. The recombinant ZIKV envelope (E) antigen can be useful for serodiagnosis of ZIKV infection and for monitoring immune responses during preclinical and clinical ZIKV vaccine development. In this study, we describe production of ZIKV E using the modified polyethyleneimine (PEI) transfection in HEK293 cells to improve cost-effective large-scale production. We show that the secretion of ZIKV E in HEK293 cells is dependent on cell culture incubation temperatures where incubation at a low temperature of 28 °C improved protein secretion of both, E-CD4 and E, whereas a substantial decrease in secretion was observed at 37 °C. The resulting E-CD4 produced at low temperature yielded similar binding profiles in ELISAs in comparison with a commercially available E protein using human seropositive sera to ZIKV. We also show that ZIKV NS1 and NS1 ß-ladder antigens produced in HEK293 cells, have similar binding profiles in ELISA which suggests that both NS1 or NS1 ß-ladder can be used for serodiagnosis of ZIKV. In conclusion, we propose a cost-effective production of the ZIKV E and NS1, suitable for both, clinical and research applications in endemic countries.

Recent advances in the production of proteins in insect and mammalian cells for structural biology.

The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.

Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains.

BACKGROUND: Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor Y. As part of a structural genomics project we have expressed single and multi-domain fragments of the different human ZHX genes for use in structure determination. RESULTS: A total of 30 single and multiple domain ZHX1-3 constructs selected from bioinformatics protocols were screened for soluble expression in E. coli using high throughput methodologies. Two homeodomains were crystallized leading to structures for ZHX1 HD4 and ZHX2 HD2. ZHX1 HD4, although closest matched to homeodomains from 'homez' and 'engrailed', showed structural differences, notably an additional C-terminal helix (helix V) which wrapped over helix I thereby making extensive contacts. Although ZHX2 HD2-3 was successfully expressed and purified, proteolysis occurred during crystallization yielding crystals of just HD2. The structure of ZHX2 HD2 showed an unusual open conformation with helix I undergoing 'domain-swapping' to form a homodimer. CONCLUSIONS: Although multiple-domain constructs of ZHX1 selected by bioinformatics studies could be expressed solubly, only single homeodomains yielded crystals. The crystal structure of ZHX1 HD4 showed additional hydrophobic interactions relative to many known homeodomains via extensive contacts formed by the novel C-terminal helix V with, in particular, helix I. Additionally, the replacement of some charged covariant residues (which are commonly observed to form salt bridges in non-homeotherms such as the Drosophila 'engrailed' homeodomain), by apolar residues further increases hydrophobic contacts within ZHX1 HD4, and potentially stability, relative to engrailed homeodomain. ZHX1 HD4 helix V points away from the normally observed DNA major groove binding site on homeodomains and thus would not obstruct the putative binding of nucleic acid. In contrast, for ZHX2 HD2 the observed altered conformation involving rearrangement of helix I, relative to the canonical homeodomain fold, disrupts the normal DNA binding site, although protein-protein binding is possible as observed in homodimer formation.

The structure of a reduced form of OxyR from Neisseria meningitidis.

BACKGROUND: Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator. OxyR responds directly to intracellular hydrogen peroxide through the reversible formation of a disulphide bond between C199 and C208 in the regulatory domain of the protein. RESULTS: We report the first crystal structure of the regulatory domain of an OxyR protein (NMB0173 from N. meningitidis) in the reduced state i.e. with cysteines at positions 199 and 208. The protein was crystallized under reducing conditions and the structure determined to a resolution of 2.4 A. The overall fold of the Neisseria OxyR shows a high degree of similarity to the structure of a C199S mutant OxyR from E. coli, which cannot form the redox sensitive disulphide. In the neisserial structure, C199 is located at the start of helix alpha3, separated by 18 A from C208, which is positioned between helices alpha3 and alpha4. In common with other LysR-type regulators, full length OxyR proteins are known to assemble into tetramers. Modelling of the full length neisserial OxyR as a tetramer indicated that C199 and C208 are located close to the dimer-dimer interface in the assembled tetramer. The formation of the C199-C208 disulphide may thus affect the quaternary structure of the protein. CONCLUSION: Given the high level of structural similarity between OxyR from N. meningitidis and E. coli, we conclude that the redox response mechanism is likely to be similar in both species, involving the reversible formation of a disulphide between C199-C208. Modelling suggests that disulphide formation would directly affect the interface between regulatory domains in an OxyR tetramer which in turn may lead to an alteration in the spacing/orientation of the DNA-binding domains and hence the interaction of OxyR with its DNA binding sites.

The structure-function relationship of oncogenic LMTK3.

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.