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The aspartate receptor is a transmembrane-signalling protein that mediates chemotaxis behaviour in bacteria. Aspartate receptors in Salmonella typhimurium and Escherichia coli exist as dimers of two subunits in the presence as well as in the absence of aspartate. We have previously reported the three-dimensional structures of the external ligand-binding domain of the S. typhimurium aspartate receptor with and without bound aspartate. The external or periplasmic region of the aspartate receptor is a dimer of four-alpha-helical bundle subunits; a single aspartate molecule binds to one of two sites residing at the subunit interface, increasing the affinity of the subunits for one another. Here we report the results of a detailed analysis of the aspartate receptor ligand-binding domain structure (residues 25 to 188). The dimer interface between the twofold related subunits consists primarily of contacts mediated by the side-chains of the N-terminal helix of each four-alpha-helical bundle subunit. The N-terminal helices pack approximately 20 degrees from parallel as an approximate coiled-coil super-secondary structure. We have refined aspartate receptor ligand-binding domain structures in the presence and in the absence of a bound aromatic compound, 1,10-phenanthroline, to 2.2 A and 2.3 A resolution, respectively, as well as crystal structures in the presence of specifically bound Au(I), Hg(II) and Pt(IV) complex ions at 2.4 A, 3.0 A and 3.3 A resolution, respectively. The possible biological relevance of the aromatic ligand-binding site and the metal ion-binding sites is discussed. The dimer of four-alpha-helical bundle subunits composing the periplasmic region of the S. typhimurium aspartate receptor provides a basis for understanding the results of mutational analyses performed on related chemotaxis transmembrane receptors. The crystal structure analysis provides an explanation for the way in which mutations in the E. coli aspartate receptor affect its binding to the periplasmic maltose-binding protein and how mutations in the more distantly related E. coli Trg chemotaxis receptor affect its binding to the periplasmic ribose and glucose-galactose binding proteins.

The three-dimensional structure of an active, disulfide cross-linked dimer of the ligand-binding domain of the Salmonella typhimurium aspartate receptor and that of an aspartate complex have been determined by x-ray crystallographic methods at 2.4 and 2.0 angstrom (A) resolution, respectively. A single subunit is a four-alpha-helix bundle with two long amino-terminal and carboxyl-terminal helices and two shorter helices that form a cylinder 20 A in diameter and more than 70 A long. The two subunits in the disulfide-bonded dimer are related by a crystallographic twofold axis in the apo structure, but by a noncrystallographic twofold axis in the aspartate complex structure. The latter structure reveals that the ligand binding site is located more than 60 A from the presumed membrane surface and is at the interface of the two subunits. Aspartate binds between two alpha helices from one subunit and one alpha helix from the other in a highly charged pocket formed by three arginines. The comparison of the apo and aspartate complex structures shows only small structural changes in the individual subunits, except for one loop region that is disordered, but the subunits appear to change orientation relative to each other. The structures of the two forms of this protein provide a step toward understanding the mechanisms of transmembrane signaling.

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3 KCTD5 ) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3 NTD /Gβ 1 γ 2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5 BTB /CUL3 NTD and KCTD5 CTD /Gβγ moieties of the structure. CRL3 KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gβ and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3 NTD /Gβγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex., Competing Interests: Competing interests statement:The authors declare no competing interest.

The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3' end of the PNT (pointed) domain, in ERG's ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG's interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention., (© 2023. Springer Nature Limited.)

B cell lymphoma 6 (BCL6), a highly regulated transcriptional repressor, is deregulated in several forms of non-Hodgkin lymphoma (NHL), most notably in diffuse large B-cell lymphoma (DLBCL). The activities of BCL6 are dependent on protein-protein interactions with transcriptional co-repressors. To find new therapeutic interventions addressing the needs of patients with DLBCL, we initiated a program to identify BCL6 inhibitors that interfere with co-repressor binding. A virtual screen hit with binding activity in the high micromolar range was optimized by structure-guided methods, resulting in a novel and highly potent inhibitor series. Further optimization resulted in the lead candidate 58 (OICR12694/JNJ-65234637), a BCL6 inhibitor with low nanomolar DLBCL cell growth inhibition and an excellent oral pharmacokinetic profile. Based on its overall favorable preclinical profile, OICR12694 is a highly potent, orally bioavailable candidate for testing BCL6 inhibition in DLBCL and other neoplasms, particularly in combination with other therapies., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI) 2 -PbI 2 ). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Ral Guanine Nucleotide Dissociation Stimulator Like 1 (RGL1) is a RAS effector protein that activates Ral GTPase by stimulating nucleotide exchange. Most structures of RAS-effector complexes are for the HRAS isoform; relatively few KRAS-effector structures have been solved, even though KRAS mutations are more frequent in human cancers. We determined crystal structures of KRAS/RGL1-RAS-association (RA) domain complexes and characterized the interaction in solution using nuclear magnetic resonance spectroscopy, size-exclusion chromatography combined with multi-angle light scattering and biolayer interferometry. We report structures of wild-type KRAS and the oncogenic G12V mutant in complex with the RA domain of RGL1 at < 2 Å resolution. KRAS WT /RGL1-RA crystallized as a 1:1 heterodimer, whilst KRAS G12V /RGL1-RA crystallized as a heterotetrameric structure in which RGL1-RA dimerized via domain-swapping the C-terminal beta-strand. Solution data indicated that KRAS WT and KRAS G12V in complex with RGL1-RA both exist predominantly as 1:1 dimers, while tetramerization occurs through very slow association. Through detailed structural analyses, the distance and angle between RAS α1 helix and RBD/RA α1 helix were found to differ significantly among RAS and RBD/RA complexes. The KRAS/RGL1-RA structures possess some of the largest α1 RAS /α1 Effector distances (21.7-22.2 Å), whereas the corresponding distances in previously reported RAS/RAF complexes are significantly shorter (15.2-17.7 Å). Contact map analysis identified unique structural signatures involving contacts between the β1-β2 loop of RA and the α1 helix of RAS, clearly distinguishing the KRAS/RGL1-RA (and other RAS/RA complexes) from RAS/RBD complexes. These results demonstrate that RAS effectors employ an assortment of finely-tuned docking surfaces to achieve optimal interactions with RAS., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL., Experimental Design: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy., Results: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment., Conclusions: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL., (©2021 American Association for Cancer Research.)

The essential eukaryotic chaperone Hsp90 regulates the form and function of diverse client proteins, many of which govern thermotolerance, virulence, and drug resistance in fungal species. However, use of Hsp90 inhibitors as antifungal therapeutics has been precluded by human host toxicities and suppression of immune responses. We recently described resorcylate aminopyrazoles (RAPs) as the first class of Hsp90 inhibitors capable of discriminating between fungal ( Cryptococcus neoformans , Candida albicans ) and human isoforms of Hsp90 in biochemical assays. Here, we report an iterative structure-property optimization toward RAPs capable of inhibiting C. neoformans growth in culture. In addition, we report the first X-ray crystal structures of C. neoformans Hsp90 nucleotide binding domain (NBD), as the apoprotein and in complexes with the non-species-selective Hsp90 inhibitor NVP-AUY922 and three RAPs revealing unique ligand-induced conformational rearrangements, which reaffirm the hypothesis that intrinsic differences in protein flexibility can confer selective inhibition of fungal versus human Hsp90 isoforms.

Fungi inhabit extraordinarily diverse ecological niches, including the human body. Invasive fungal infections have a devastating impact on human health worldwide, killing ∼1.5 million individuals annually. The majority of these deaths are attributable to species of Candida , Cryptococcus, and Aspergillus Treating fungal infections is challenging, in part due to the emergence of resistance to our limited arsenal of antifungal agents, necessitating the development of novel therapeutic options. Whereas conventional antifungal strategies target proteins or cellular components essential for fungal growth, an attractive alternative strategy involves targeting proteins that regulate fungal virulence or antifungal drug resistance, such as regulators of fungal stress responses. Stress response networks enable fungi to adapt, grow, and cause disease in humans and include regulators that are highly conserved across eukaryotes as well as those that are fungal-specific. This review highlights recent developments in elucidating crystal structures of fungal stress response regulators and emphasizes how this knowledge can guide the design of fungal-selective inhibitors. We focus on the progress that has been made with highly conserved regulators, including the molecular chaperone Hsp90, the protein phosphatase calcineurin, and the small GTPase Ras1, as well as with divergent stress response regulators, including the cell wall kinase Yck2 and trehalose synthases. Exploring structures of these important fungal stress regulators will accelerate the design of selective antifungals that can be deployed to combat life-threatening fungal diseases., Competing Interests: Conflict of interest—L. E. C. is a co-founder and shareholder in Bright Angel Therapeutics, a platform company for development of novel antifungal therapeutics. L. E. C. is a consultant for Boragen, a small-molecule development company focused on leveraging the unique chemical properties of boron chemistry for crop protection and animal health., (© 2020 LeBlanc et al.)

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system., Competing Interests: Declaration of Interests A.M.M. receives research funding for Janssen, is on the scientific board of KDAC Pharmaceuticals, and has consulted for Constellation and Epizyme. M.R.G. consults for Verastem Oncology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Adipokines secreted from white adipose tissue play a role in metabolic crosstalk and homeostasis, whereas the brown adipose secretome is less explored. We performed high-sensitivity mass-spectrometry-based proteomics on the cell media of human adipocytes derived from the supraclavicular brown adipose and from the subcutaneous white adipose depots of adult humans. We identified 471 potentially secreted proteins covering interesting categories such as hormones, growth factors, extracellular matrix proteins, and proteins of the complement system, which were differentially regulated between brown and white adipocytes. A total of 101 proteins were exclusively quantified in brown adipocytes, and among these was ependymin-related protein 1 (EPDR1). EPDR1 was detected in human plasma, and functional studies suggested a role for EPDR1 in thermogenic determination during adipogenesis. In conclusion, we report substantial differences between the secretomes of brown and white human adipocytes and identify novel candidate batokines that can be important regulators of human metabolism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol - like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.

Several lines of evidence link the canonical oncogene BCL6 to stress response. Here we demonstrate that BCL6 evolved in vertebrates as a component of the HSF1-driven stress response, which has been co-opted by the immune system to support germinal center formation and may have been decisive in the convergent evolution of humoral immunity in jawless and jawed vertebrates. We find that the highly conserved BTB corepressor binding site of BCL6 mediates stress adaptation across vertebrates. We demonstrate that pan-cancer cells hijack this stress tolerance mechanism to aberrantly express BCL6. Targeting the BCL6 BTB domain in cancer cells induces apoptosis and increases susceptibility to repeated doses of cytotoxic therapy. The chemosensitization effect upon BCL6 BTB inhibition is dependent on the derepression of TOX , implicating modulation of DNA repair as a downstream mechanism. Collectively, these data suggest a form of adaptive nononcogene addiction rooted in the natural selection of BCL6 during vertebrate evolution. SIGNIFICANCE: We demonstrate that HSF1 drives BCL6 expression to enable stress tolerance in vertebrates. We identify an HSF1-BCL6-TOX stress axis that is required by cancer cells to tolerate exposure to cytotoxic agents and points toward BCL6-targeted therapy as a way to more effectively kill a wide variety of solid tumors. This article is highlighted in the In This Issue feature, p. 565 ., (©2019 American Association for Cancer Research.)

EPDR1, a member of the ependymin-related protein family, is a relatively uncharacterized protein found in the lysosomes and secretomes of most vertebrates. Despite having roles in human disease and health, the molecular functions of EPDR1 remain unknown. Here, we present crystal structures of human EPDR1 and reveal that the protein adopts a fold previously seen only in bacterial proteins related to the LolA lipoprotein transporter. EPDR1 forms a homodimer with an overall shape resembling a half-shell with two non-overlapping hydrophobic grooves on the flat side of the hemisphere. EPDR1 can interact with membranes that contain negatively charged lipids, including BMP and GM1, and we suggest that EPDR1 may function as a lysosomal activator protein or a lipid transporter. A phylogenetic analysis reveals that the fold is more widely distributed than previously suspected, with representatives identified in all branches of cellular life., Competing Interests: The authors declare no competing interests.

Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs ( PL PDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB 5 ). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the PL PDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the PL PDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB 5 measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB 5 was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB 5 measured for pre-loaded PDs and PL PDs prepared from glycolipids extracted from pig and mouse brain revealed that the PL PDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest PL PDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS. Graphical Abstract ᅟ.

The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion., (Copyright © 2017 American Society for Microbiology.)

Saposin A (SapA) lipoprotein discs, also known as picodiscs (PDs), represent an attractive method to solubilize glycolipids for protein interaction studies in aqueous solution. Recent electrospray ionization mass spectrometry (ESI-MS) data suggest that the size and composition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-containing PDs at neutral pH differs from those of N,N-dimethyldodecylamine N-oxide determined by X-ray crystallography. Using high-resolution ESI-MS, multiangle laser light scattering (MALLS), and molecular dynamics (MD) simulations, the composition, heterogeneity, and structure of POPC-PDs in aqueous ammonium acetate solutions at pH 4.8 and 6.8 were investigated. The ESI-MS and MALLS data revealed that POPC-PDs consist predominantly of (SapA dimer + iPOPC) complexes, with i = 23-29, and have an average molecular weight (MW) of 38.2 ± 3.3 kDa at pH 4.8. In contrast, in freshly prepared solutions at pH 6.8, POPC-PDs are composed predominantly of (SapA tetramer + iPOPC) complexes, with i = 37-60, with an average MW of 68.0 ± 2.7 kDa. However, the (SapA tetramer + iPOPC) complexes are unstable at neutral pH and convert, over a period of hours, to (SapA trimer + iPOPC) complexes, with i = 29-36, with an average MW of 51.1 ± 2.9 kDa. The results of molecular modeling suggest spheroidal structures for the (SapA dimer + iPOPC), (SapA trimer + iPOPC), and (SapA tetramer + iPOPC) complexes in solution. Comparison of measured collision cross sections (Ω) with values calculated for gaseous (SapA dimer + 26POPC) 8+ , (SapA trimer + 33POPC) 12+ , and (SapA tetramer + 42POPC) 16+ ions produced from modeling suggests that the solution structures are largely preserved in the gas phase, although the lipids do not maintain regular bilayer orientations.

Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Membrane-less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher-order assemblies influences the recruitment of the speckle-type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin-3-RING ubiquitin ligase (CRL3) substrate adaptor, self-associates into higher-order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild-type SPOP localizes to liquid nuclear speckles, self-association-deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB-mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration-dependent populations of the resulting oligomeric species. Higher-order oligomerization of SPOP stimulates CRL3(SPOP) ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher-order protein self-association may be a general mechanism to concentrate functional components in membrane-less cellular bodies., (© 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

This work describes the application of the catch-and-release electrospray ionization-mass spectrometry (CaR-ESI-MS) assay, implemented using picodiscs (complexes comprised of saposin A and lipids, PDs), to screen mixtures of glycolipids (GLs) against water-soluble proteins to detect specific interactions. To demonstrate the reliability of the method, seven gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD2, and GT1b) were incorporated, either individually or as a mixture, into PDs and screened against two lectins: the B subunit homopentamer of cholera toxin (CTB5) and a subfragment of toxin A from Clostridium difficile (TcdA-A2). The CaR-ESI-MS results revealed that CTB5 binds to six of the gangliosides (GM1, GM2, GM3, GD1a, GD1b, and GT1b), while TcdA-A2 binds to five of them (GM1, GM2, GM3, GD1a, and GT1b). These findings are consistent with the measured binding specificities of these proteins for ganglioside oligosaccharides. Screening mixtures of lipids extracted from porcine brain and a human epithelial cell line against CTB5 revealed binding to multiple GM1 isoforms as well as to fucosyl-GM1, which is a known ligand. Finally, a comparison of the present results with data obtained with the CaR-ESI-MS assay implemented using nanodiscs (NDs) revealed that the PDs exhibited similar or superior performance to NDs for protein-GL binding measurements.

Cullin3 (Cul3)-based ubiquitin E3 ligase complexes catalyze the transfer of ubiquitin from an E2 enzyme to target substrate proteins. In these assemblies, the C-terminal region of Cul3 binds Rbx1/E2-ubiquitin, while the N-terminal region interacts with various BTB (bric-à-brac, tramtrack, broad complex) domain proteins that serve as substrate adaptors. Previous crystal structures of the homodimeric BTB proteins KLHL3, KLHL11 and SPOP in complex with the N-terminal domain of Cul3 revealed the features required for Cul3 recognition in these proteins. A second class of BTB-domain-containing proteins, the KCTD proteins, is also Cul3 substrate adaptors, but these do not share many of the previously identified determinants for Cul3 binding. We report the pentameric crystal structures of the KCTD1 and KCTD9 BTB domains and identify plasticity in the KCTD1 rings. We find that the KCTD proteins 5, 6, 9 and 17 bind to Cul3 with high affinity, while the KCTD proteins 1 and 16 do not have detectable binding. Finally, we confirm the 5:5 assembly of KCTD9/Cul3 complexes by cryo-electron microscopy and provide a molecular rationale for BTB-mediated Cul3 binding specificity in the KCTD family., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Escherichia Coli GnsA is a regulator of phosphatidylethanolamine synthesis and functions as a suppressor of both a secG null mutation and fabA6 mutations. GnsA may also be a toxin with the cognate antitoxin YmcE. Here we report the crystal structure of GnsA to 1.8 Å. GnsA forms a V shaped hairpin structure that is tightly associated into a homodimer. Our comprehensive structural study suggests that GnsA is structurally similar to an outer membrane protein, suggesting a function of protein binding., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Protein interactions with glycolipids are implicated in diverse cellular processes. However, the study of protein-glycolipid complexes remains a significant experimental challenge. Here, we describe a powerful new assay that combines electrospray ionization mass spectrometry (ESI-MS) and picodiscs, which are composed of human sphingolipid activator protein saposin A and a small number of phospholipids, to display glycolipids in a lipid environment for protein-glycolipid interaction studies in aqueous solution. Time-resolved measurements of enzyme catalyzed hydrolysis of glycolipid substrates and the detection of low, moderate, and high affinity protein-glycolipid interactions serve to demonstrate the reliability and versatility of the assay.

Protein kinase A (PKA) hyperactivation causes hereditary endocrine neoplasias; however, its role in sporadic epithelial cancers is unknown. Here, we show that heightened PKA activity in the mammary epithelium generates tumors. Mammary-restricted biallelic ablation of Prkar1a, which encodes for the critical type-I PKA regulatory subunit, induced spontaneous breast tumors characterized by enhanced type-II PKA activity. Downstream of this, Src phosphorylation occurs at residues serine-17 and tyrosine-416 and mammary cell transformation is driven through a mechanism involving Src signaling. The phenotypic consequences of these alterations consisted of increased cell proliferation and, accordingly, expansion of both luminal and basal epithelial cell populations. In human breast cancer, low PRKAR1A/high SRC expression defines basal-like and HER2 breast tumors associated with poor clinical outcome. Together, the results of this study define a novel molecular mechanism altered in breast carcinogenesis and highlight the potential strategy of inhibiting SRC signaling in treating this cancer subtype in humans.

Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.

Farber disease (FD) is a severe inherited disorder of lipid metabolism characterized by deficient lysosomal acid ceramidase (ACDase) activity, resulting in ceramide accumulation. Ceramide and metabolites have roles in cell apoptosis and proliferation. We introduced a single-nucleotide mutation identified in human FD patients into the murine Asah1 gene to generate the first model of systemic ACDase deficiency. Homozygous Asah1(P361R/P361R) animals showed ACDase defects, accumulated ceramide, demonstrated FD manifestations and died within 7-13 weeks. Mechanistically, MCP-1 levels were increased and tissues were replete with lipid-laden macrophages. Treatment of neonates with a single injection of human ACDase-encoding lentivector diminished the severity of the disease as highlighted by enhanced growth, decreased ceramide, lessened cellular infiltrations and increased lifespans. This model of ACDase deficiency offers insights into the pathophysiology of FD and the roles of ACDase, ceramide and related sphingolipids in cell signaling and growth, as well as facilitates the development of therapy., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85 and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600 mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

KLHL3 is a BTB-BACK-Kelch family protein that serves as a substrate adapter in Cullin3 (Cul3) E3 ubiquitin ligase complexes. KLHL3 is highly expressed in distal nephron tubules where it is involved in the regulation of electrolyte homeostasis and blood pressure. Mutations in KLHL3 have been identified in patients with inherited hypertension disorders, and several of the disease-associated mutations are located in the presumed Cul3 binding region. Here, we report the crystal structure of a complex between the KLHL3 BTB-BACK domain dimer and two copies of an N terminal fragment of Cul3. We use isothermal titration calorimetry to directly demonstrate that several of the disease mutations in the KLHL3 BTB-BACK domains disrupt the association with Cul3. Both the BTB and BACK domains contribute to the Cul3 interaction surface, and an extended model of the dimeric CRL3 complex places the two E2 binding sites in a suprafacial arrangement with respect to the presumed substrate-binding sites.

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4 Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The saposins are small, membrane-active proteins that exist in both soluble and lipid-bound states. Saposin A has roles in sphingolipid catabolism and transport and is required for the breakdown of galactosylceramide by β-galactosylceramidase. In the absence of lipid, saposin A adopts a closed monomeric apo conformation typical of this family. To study a lipid-bound state of this protein, we determined the crystal structure of saposin A in the presence of detergent to 1.9 Å resolution. The structure reveals two chains of saposin A in an open conformation encapsulating 40 internally bound detergent molecules organized in a highly ordered bilayer-like hydrophobic core. The complex provides a high-resolution view of a discoidal lipoprotein particle in which all of the internalized acyl chains are resolved. Saposin A lipoprotein discs exhibit limited selectivity with respect to the incorporated lipid, and can solubilize phospholipids, sphingolipids, and cholesterol into discrete, monodisperse particles with mass of approximately 27 kDa. These discs may be the smallest possible lipoprotein structures that are stabilized by lipid self-assembly.

Enzymatic reactions involving bilayer lipids occur in an environment with strict physical and topological constraints. The integral membrane enzyme PagP transfers a palmitoyl group from a phospholipid to lipid A in order to assist Escherichia coli in evading host immune defenses during infection. PagP measures the palmitoyl group with an internal hydrocarbon ruler that is formed in the interior of the eight-stranded antiparallel β barrel. The access and egress of the palmitoyl group is thought to take a lateral route from the bilayer phase to the barrel interior. Molecular dynamics, mutagenesis, and a 1.4 A crystal structure of PagP in an SDS / 2-methyl-2,4-pentanediol (MPD) cosolvent system reveal that phospholipid access occurs at the crenel present between strands F and G of PagP. In this way, the phospholipid head group can remain exposed to the cell exterior while the lipid acyl chain remains in a predominantly hydrophobic environment as it translocates to the protein interior., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The BTB domain is a widely distributed protein-protein interaction motif that is often found at the N-terminus of zinc finger transcription factors. Previous crystal structures of BTB domains have revealed tightly interwound homodimers, with the N-terminus from one chain forming a two-stranded anti-parallel beta-sheet with a strand from the other chain. We have solved the crystal structures of the BTB domains from Fanconi anemia zinc finger (FAZF) and Miz1 (Myc-interacting zinc finger 1) to resolutions of 2.0 A and 2.6 A, respectively. Unlike previous examples of BTB domain structures, the FAZF BTB domain is a nonswapped dimer, with each N-terminal beta-strand associated with its own chain. As a result, the dimerization interface in the FAZF BTB domain is about half as large as in the domain-swapped dimers. The Miz1 BTB domain resembles a typical swapped BTB dimer, although it has a shorter N-terminus that is not able to form the interchain sheet. Using cysteine cross-linking, we confirmed that the promyelocytic leukemia zinc finger (PLZF) BTB dimer is strand exchanged in solution, while the FAZF BTB dimer is not. A phylogenic tree of the BTB fold based on both sequence and structural features shows that the common ancestor of the BTB domain in BTB-ZF (bric à brac, tramtrack, broad-complex zinc finger) proteins was a domain-swapped dimer. The differences in the N-termini seen in the FAZF and Miz1 BTB domains appear to be more recent developments in the structural evolution of the domain., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Distal myopathies are a heterogeneous group of disorders characterized by progressive weakness and muscular atrophy, beginning in distal limb muscles and affecting proximal limb muscles at a later stage. We studied a large German kindred with 10 affected members. Weakness and atrophy of the anterior tibial muscles started between the ages of 8 and 16 years, followed by atrophy of intrinsic hand muscles. Progression was slow, and patients retained the ability to walk until the seventh decade. Serum creatinine kinase levels were increased in the range of 150-1400 U/l. Muscle biopsies showed myopathic changes, whereas immunohistochemistry showed normal expression of marker proteins for muscular dystrophies. Patients had reduced sensation with stocking-glove distribution in the distal limbs in later life. Nerve conduction studies revealed no evidence of neuropathy. Genome-wide linkage analysis in this family revealed a new locus for distal myopathy at 9p21.2-p22.3 (multipoint logarithm of the odds ratio=4.21). By positional cloning we found a heterozygous mutation L95F in the Kelch-like homologue 9 gene, encoding a bric-a-brac Kelch protein. Molecular modelling indicated that the mutation may interfere with the interaction of the bric-a-brac domain with Cullin 3. Coimmunoprecipitation experiments confirmed that the mutation reduces association with Cullin 3 in the Kelch-like homologue 9-Cullin 3-E3 ubiquitin ligase complex, which is involved in ubiquitin-dependent protein degradation. We identified a unique form of early onset autosomal dominant distal myopathy which is associated with a Kelch-like homologue 9 mutation and interferes with normal skeletal muscle through a novel pathogenetic mechanism.

The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients., (Copyright 2010 Elsevier Inc. All rights reserved.)

Protein stabilization upon ligand binding has frequently been used to identify ligands for soluble proteins. Methods such as differential scanning fluorimetry (DSF) and differential static light scattering (DSLS) have been employed in the 384-well format and have been useful in identifying ligands that promote crystallization and 3D structure determination of proteins. However, finding a generic method that is applicable to membrane proteins has been a challenge as the high hydrophobicity of membrane proteins and the presence of detergents essential for their solubilization interfere with fluorescence-based detections. Here the authors used MsbA (an adenosine triphosphate binding cassette transporter), CorA (a Mg(++) channel), and CpxA (a histidine kinase) as model proteins and show that DSLS is not sensitive to the presence of detergents or protein hydrophobicity and can be used to monitor thermodenaturation of membrane proteins, assess their stability, and detect ligand binding in a 384-well format.

An ideal detergent would be able to maintain a membrane protein in a soluble state with no measurable effect on the functional, structural, and thermodynamic properties of the protein relative to the bilayer-embedded state. Unfortunately, the detergents that are commonly used by membrane protein biochemists fall short of this standard. Although remarkable advances have been made in membrane protein structural biology, there remains a need for improved detergents that provide a more natural substitute for the membrane environment. Lipopeptide detergents (LPDs) are a new class of amphiphile designed to be better mimics of the bilayer at the hydrophobic surfaces of solubilized membrane proteins. LPDs consist of an alpha-helical peptide backbone that supports alkyl chains anchored at either end of the helix. The LPD monomers self-assemble into cylindrical micelles with a membrane-like packing of the inner core of alkyl chains.

Lipopeptide detergents (LPDs) are a new class of amphiphile designed specifically for the structural study of integral membrane proteins. The LPD monomer consists of a 25-residue peptide with fatty acyl chains linked to side chains located at positions 2 and 24 of the peptide. LPDs are designed to form alpha-helices that self-assemble into cylindrical micelles, providing a more natural interior acyl chain packing environment relative to traditional detergents. We have determined the crystal structure of LPD-12, an LPD coupled to two dodecanoic acids, to a resolution of 1.20 A. The LPD-12 monomers adopt the target conformation and associate into cylindrical octamers as expected. Pairs of helices are strongly associated as Alacoil-type antiparallel dimers, and four of these dimers interact through much looser contacts into assemblies with approximate D(2) symmetry. The aligned helices form a cylindrical shell with a hydrophilic exterior that protects an interior hydrophobic cavity containing the 16 LPD acyl chains. Over 90% of the methylene/methyl groups from the acylated side chains are visible in the micelle interiors, and approximately 90% of these adopt trans dihedral angle conformations. Dodecylmaltoside (DDM) was required for the crystallization of LPD-12, and we find 10-24 ordered DDM molecules associated with each LPD assembly, resulting in an overall micelle molecular weight of approximately 30 kDa. The structures confirm the major design objectives of the LPD framework, and reveal unexpected features that will be helpful in the engineering additional versions of lipopeptide amphiphiles.

Background: Recently, we reported a unique approach to preserve the activity of some proteins in the presence of the denaturing agent, Sodium Dodecyl Sulfate (SDS). This was made possible by addition of the amphipathic solvent 2,4-Methyl-2-PentaneDiol (MPD), used as protecting but also as refolding agent for these proteins. Although the persistence of the protein activity in the SDS/MPD mixture was clearly established, preservation of their structure was only speculative until now., Results: In this paper, a detailed X-ray study addresses the pending question. Crystals of hen egg-white lysozyme were grown for the first time in the presence of MPD and denaturing concentrations of SDS. Depending on crystallization conditions, tetragonal crystals in complex with either SDS or MPD were collected. The conformation of both structures was very similar to the native lysozyme and the obtained complexes of SDS-lysozyme and MPD-lysozyme give some insights in the interplay of protein-SDS and protein-MPD interactions., Conclusion: This study clearly established the preservation of the enzyme structure in a SDS/MPD mixture. It is hypothesized that high concentrations of MPD would change the properties of SDS and lower or avoid interactions between the denaturant and the protein. These structural data therefore support the hypothesis that MPD avoids disruption of the enzyme structure by SDS and can protect proteins from SDS denaturation.

Saposin D is a sphingolipid activator protein required for the lysosomal breakdown of ceramide to a fatty acid and sphingosine by acid ceramidase. The crystal structure of saposin D has been determined in two different crystal forms, resulting in a total of six crystallographically independent views of this small 80-amino-acid protein. All of the structures are highly similar and reveal the monomeric form of the saposin fold previously seen in the crystal structures of saposins A and C. Saposin D is slightly more compact than the related saposins A and C owing to a slight repositioning of the 'stem' and 'hairpin' regions of the protein.

The transcriptional corepressors BCOR, SMRT, and NCoR are known to bind competitively to the BCL6 BTB domain despite the fact that BCOR has no detectable sequence similarity to the other two corepressors. We have identified a 17 residue motif from BCOR that binds directly to the BCL6 BTB domain and determined the crystal structure of the complex to a resolution of 2.6 A. Remarkably, the BCOR BCL6 binding domain (BCOR(BBD)) peptide binds in the same BCL6 binding site as the SMRT(BBD) peptide despite the lack of any significant sequence similarity between the two peptides. Mutations of critical BCOR(BBD) residues cause the disruption of the BCL6 corepression activities of BCOR, and a BCOR(BBD) peptide blocks BCL6-mediated transcriptional repression and kills lymphoma cells.

Sodium dodecyl sulfate (SDS) is a highly effective and widely used protein denaturant. We show that certain amphipathic cosolvents such as 2-methyl-2,4-pentanediol (MPD) can protect proteins from SDS denaturation, and in several cases can refold proteins from the SDS-denatured state. This cosolvent effect is observed with integral membrane proteins and soluble proteins from either the alpha-helical or the beta-sheet structural classes. The SDS/MPD system can be used to study processes involving native protein states, and we demonstrate the reversible thermal denaturation of the outer membrane protein PagP in an SDS/MPD buffer. MPD and related cosolvents can modulate the denaturing properties of SDS, and we describe a simple and effective method to recover refolded, active protein from the SDS-denatured state.

Acid beta-glucosidase (GCase) is a soluble lysosomal enzyme responsible for the hydrolysis of glucose from glucosylceramide and requires activation by the small nonenzymatic protein saposin C (sapC) to gain access to the membrane-embedded glycosphingolipid substrate. We have used in situ atomic force microscopy (AFM) with simultaneous confocal and epifluorescence microscopies to investigate the interactions of GCase and sapC with lipid bilayers. GCase binds to sites on membranes transformed by sapC, and enzyme activity occurs at loci containing both GCase and sapC. Using FRET, we establish the presence of GCase/sapC and GCase/product contacts in the bilayer. These data support a mechanism in which sapC locally alters regions of bilayer for subsequent attack by the enzyme in stably bound protein complexes.

The structural basis of lipid acyl-chain selection by membrane-intrinsic enzymes is poorly understood because most integral membrane enzymes of lipid metabolism have proven refractory to structure determination; however, robust enzymes from the outer membranes of gram-negative bacteria are now providing a first glimpse at the underlying mechanisms. The methylene unit resolution of the phospholipid:lipid A palmitoyltransferase PagP is determined by the hydrocarbon ruler, a 16-carbon saturated acyl-chain-binding pocket buried within the transmembrane beta-barrel structure. Substitution of Gly88 lining the floor of the hydrocarbon ruler with Ala or Met makes the enzyme select specifically 15- or 12-carbon saturated acyl chains, respectively, indicating that hydrocarbon ruler depth determines acyl-chain selection. However, the Gly88Cys PagP resolution does not diminish linearly because it selects both 14- and 15-carbon saturated acyl chains. We discovered that an exciton, emanating from a buried Tyr26-Trp66 phenol-indole interaction, is extinguished by a local structural perturbation arising from the proximal Gly88Cys PagP sulfhydryl group. Site-specific S-methylation of the single Cys afforded Gly88Cys-S-methyl PagP, which reasserted both the exciton and methylene unit resolution by specifically selecting 13-carbon saturated acyl chains for transfer to lipid A. Unlike the other Gly88 substitutions, the Cys sulfhydryl group recedes from the hydrocarbon ruler floor and locally perturbs the subjacent Tyr26 and Trp66 aromatic rings. The resulting hydrocarbon ruler expansion thus occurs at the exciton's expense and accommodates an extra methylene unit in the selected acyl chain. The hydrocarbon ruler-exciton juxtaposition endows PagP with a molecular gauge for probing the structural basis of lipid acyl-chain selection in a membrane-intrinsic environment.

The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but as a result, they have complex properties in solution. It can be difficult to maintain a solubilized membrane protein in a native conformational state, and the non-specific aggregation of detergent-solubilized proteins is a common problem. Empirical "stability screens" can be helpful in choosing which detergents, and which detergent concentrations, may be optimal for a given system.

BTB-zinc finger (BTB-ZF) proteins are transcription regulators with roles in development, differentiation, and oncogenesis. In these proteins, the BTB domain (also known as the POZ domain) is a protein-protein interaction motif that contains a dimerization interface, a possible oligomerization surface, and surfaces for interactions with other factors, including nuclear co-repressors and histone deacetylases. The BTB-ZF protein LRF (also known as ZBTB7, FBI-1, OCZF, and Pokemon) is a master regulator of oncogenesis, and represses the transcription of a variety of important genes, including the ARF, c-fos, and c-myc oncogenes and extracellular matrix genes. We determined the crystal structure of the BTB domain from human LRF to 2.1 A and observed the canonical BTB homodimer fold. However, novel features are apparent on the surface of the homodimer, including differences in the lateral groove and charged pocket regions. The residues that line the lateral groove have little similarity with the equivalent residues from the BCL6 BTB domain, and we show that the 17-residue BCL6 Binding Domain (BBD) from the SMRT co-repressor does not bind to the LRF BTB domain.

Green fluorescent protein (GFP) and its relatives (GFP protein family) have been isolated from marine organisms such as jellyfish and corals that belong to the phylum Cnidaria (stinging aquatic invertebrates). They are intrinsically fluorescent proteins. In search of new members of the family of green fluorescent protein family, we identified a non-fluorescent chromoprotein from the Cnidopus japonicus species of sea anemone that possesses 45% sequence identity to dsRed (a red fluorescent protein). This newly identified blue color protein has an absorbance maximum of 610 nm and is hereafter referred to as cjBlue. Determination of the cjBlue 1.8 A crystal structure revealed a chromophore comprised of Gln(63)-Tyr(64)-Gly(65). The ring stacking between Tyr(64) and His(197) stabilized the cjBlue trans chromophore conformation along the Calpha2-Cbeta2 bond of 5-[(4-hydroxyphenyl)methylene]-imidazolinone, which closely resembled that of the "Kindling Fluorescent Protein" and Rtms5. Replacement of Tyr(64) with Leu in wild-type cjBlue produced a visible color change from blue to yellow with a new absorbance maximum of 417 nm. Interestingly, the crystal structure of the yellow mutant Y64L revealed two His(197) imidazole ring orientations, suggesting a flip-flop interconversion between the two conformations in solution. We conclude that the dynamics and structure of the chromophore are both essential for the optical appearance of these color proteins.

Saposins A, B, C and D are soluble, non-enzymatic proteins that interact with lysosomal membranes to activate the breakdown and transfer of glycosphingolipids. The mechanisms of hydrolase activation and lipid transfer by saposins remain unknown. We have used in situ atomic force microscopy (AFM) with simultaneous confocal fluorescence microscopy to investigate the interactions of saposins with lipid membranes. AFM images of the effect of saposins A, B and C on supported lipid bilayers showed a time and concentration-dependent nucleated spread of membrane transformation. Saposin B produced deep gaps that ultimately filled with granular material, while saposins A and C lead to localized areas of membrane that were reduced in height by approximately 1.5 nm. Fluorescence-labeled saposin C co-localized with the transformed areas of the bilayer, indicating stable binding to the membrane. Fluorescence resonance energy transfer confirmed a direct interaction between saposin C and lipid. Under certain conditions of membrane lipid composition and saposin concentration, extensive bilayer lipid removal was observed. We propose a multi-step mechanism that integrates the structural features and amphipathic properties of the saposin proteins.