Trans-activating mutations of the pseudokinase ERBB3.

Genetic changes in the ERBB family of receptor tyrosine kinases serve as oncogenic driver events and predictive biomarkers for ERBB inhibitor drugs. ERBB3 is a pseudokinase member of the family that, although lacking a fully active kinase domain, is well known for its potent signaling activity as a heterodimeric complex with ERBB2. Previous studies have identified few transforming ERBB3 mutations while the great majority of the hundreds of different somatic ERBB3 variants observed in different cancer types remain of unknown significance. Here, we describe an unbiased functional genetics screen of the transforming potential of thousands of ERBB3 mutations in parallel. The screen based on a previously described iSCREAM (in vitro screen of activating mutations) platform, and addressing ERBB3 pseudokinase signaling in a context of ERBB3/ERBB2 heterodimers, identified 18 hit mutations. Validation experiments in Ba/F3, NIH 3T3, and MCF10A cell backgrounds demonstrated the presence of both previously known and unknown transforming ERBB3 missense mutations functioning either as single variants or in cis as a pairwise combination. Drug sensitivity assays with trastuzumab, pertuzumab and neratinib indicated actionability of the transforming ERBB3 variants.

Comprehensive characterization of the embryonic factor LEUTX.

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.

DUX4 is a multifunctional factor priming human embryonic genome activation.

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

Immunogenic SARS-CoV-2 Epitopes: In Silico Study Towards Better Understanding of COVID-19 Disease-Paving the Way for Vaccine Development.

The emergence of the COVID-19 outbreak at the end of 2019, caused by the novel coronavirus SARS-CoV-2, has, to date, led to over 13.6 million infections and nearly 600,000 deaths. Consequently, there is an urgent need to better understand the molecular factors triggering immune defense against the virus and to develop countermeasures to hinder its spread. Using in silico analyses, we showed that human major histocompatibility complex (MHC) class I cell-surface molecules vary in their capacity for binding different SARS-CoV-2-derived epitopes, i.e., short sequences of 8-11 amino acids, and pinpointed five specific SARS-CoV-2 epitopes that are likely to be presented to cytotoxic T-cells and hence activate immune responses. The identified epitopes, each one of nine amino acids, have high sequence similarity to the equivalent epitopes of SARS-CoV virus, which are known to elicit an effective T cell response in vitro. Moreover, we give a structural explanation for the binding of SARS-CoV-2-epitopes to MHC molecules. Our data can help us to better understand the differences in outcomes of COVID-19 patients and may aid the development of vaccines against SARS-CoV-2 and possible future outbreaks of novel coronaviruses.

Structural and functional characteristics of xenavidin, the first frog avidin from Xenopus tropicalis.

BACKGROUND: Avidins are proteins with extraordinarily high ligand-binding affinity, a property which is used in a wide array of life science applications. Even though useful for biotechnology and nanotechnology, the biological function of avidins is not fully understood. Here we structurally and functionally characterise a novel avidin named xenavidin, which is to our knowledge the first reported avidin from a frog. RESULTS: Xenavidin was identified from an EST sequence database for Xenopus tropicalis and produced in insect cells using a baculovirus expression system. The recombinant xenavidin was found to be homotetrameric based on gel filtration analysis. Biacore sensor analysis, fluorescently labelled biotin and radioactive biotin were used to evaluate the biotin-binding properties of xenavidin - it binds biotin with high affinity though less tightly than do chicken avidin and bacterial streptavidin. X-ray crystallography revealed structural conservation around the ligand-binding site, while some of the loop regions have a unique design. The location of structural water molecules at the entrance and/or within the ligand-binding site may have a role in determining the characteristic biotin-binding properties of xenavidin. CONCLUSION: The novel data reported here provide information about the biochemically and structurally important determinants of biotin binding. This information may facilitate the discovery of novel tools for biotechnology.

Molecular features of steroid-binding antidins and their use for assaying serum progesterone.

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10-15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.

Rational modification of ligand-binding preference of avidin by circular permutation and mutagenesis.

Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4-->3) that lacks the loop between beta-strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4'-hydroxyazobenzene-2-carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4-->3 even further, we mutated asparagine 118 on the bottom of the ligand-binding pocket to methionine, which simultaneously caused a significant drop in biotin-binding affinity. The X-ray structure of cpAvd4--> 3(N118M) allows an understanding of the effect of mutation to biotin-binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion-fold between wild-type avidin and cpAvd4-->3(N118M). To demonstrate the versatility of the cpAvd4-->3 construct, we have shown that it is possible to link cpAvd4-->3 and cpAvd5-->4 to form the dual-chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self-organising nanoscale avidin building blocks.

Phylogenetic and mutational analyses of human LEUTX, a homeobox gene implicated in embryogenesis.

Recently, human PAIRED-LIKE homeobox transcription factor (TF) genes were discovered whose expression is limited to the period of embryo genome activation up to the 8-cell stage. One of these TFs is LEUTX, but its importance for human embryogenesis is still subject to debate. We confirmed that human LEUTX acts as a TAATCC-targeting transcriptional activator, like other K50-type PAIRED-LIKE TFs. Phylogenetic comparisons revealed that Leutx proteins are conserved across Placentalia and comprise two conserved domains, the homeodomain, and a Leutx-specific domain containing putative transcriptional activation motifs (9aaTAD). Examination of human genotype resources revealed 116 allelic variants in LEUTX. Twenty-four variants potentially affect function, but they occur only heterozygously at low frequency. One variant affects a DNA-specificity determining residue, mutationally reachable by a one-base transition. In vitro and in silico experiments showed that this LEUTX mutation (alanine to valine at position 54 in the homeodomain) results in a transactivational loss-of-function to a minimal TAATCC-containing promoter and a 36 bp motif enriched in genes involved in embryo genome activation. A compensatory change in residue 47 restores function. The results support the notion that human LEUTX functions as a transcriptional activator important for human embryogenesis.

Structural evidence for adaptive ligand binding of glycolipid transfer protein.

Glycolipids participate in many important cellular processes and they are bound and transferred with high specificity by glycolipid transfer protein (GLTP). We have solved three different X-ray structures of bovine GLTP at 1.4 angstroms, 1.6 angstroms and 1.8 angstroms resolution, all with a bound fatty acid or glycolipid. The 1.4 angstroms structure resembles the recently characterized apo-form of the human GLTP but the other two structures represent an intermediate conformation of the apo-GLTPs and the human lactosylceramide-bound GLTP structure. These novel structures give insight into the mechanism of lipid binding and how GLTP may conformationally adapt to different lipids. Furthermore, based on the structural comparison of the GLTP structures and the three-dimensional models of the related Podospora anserina HET-C2 and Arabidopsis thaliana accelerated cell death protein, ACD11, we give structural explanations for their specific lipid binding properties.

Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.

Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilised the protein. The modified dcAvd forms were found to retain their characteristic pseudotetrameric state both at high and low pH, and were shown to bind D-biotin at levels comparable to that of wild-type chicken avidin. The crystal structure of dcAvd-biotin complex at 1.95 Angstroms resolution demonstrates the formation of the functional dcAvd pseudotetramer at the atomic level and reveals the molecular basis for its special properties. Altogether, our data facilitate further engineering of the biotechnologically valuable dcAvd scaffold and gives insights into how to guide the quaternary structure assembly of oligomeric proteins.

Structure and characterization of a novel chicken biotin-binding protein A (BBP-A).

BACKGROUND: The chicken genome contains a BBP-A gene showing similar characteristics to avidin family genes. In a previous study we reported that the BBP-A gene may encode a biotin-binding protein due to the high sequence similarity with chicken avidin, especially at regions encoding residues known to be located at the ligand-binding site of avidin. RESULTS: Here, we expand the repertoire of known macromolecular biotin binders by reporting a novel biotin-binding protein A (BBP-A) from chicken. The BBP-A recombinant protein was expressed using two different expression systems and purified with affinity chromatography, biochemically characterized and two X-ray structures were solved - in complex with D-biotin (BTN) and in complex with D-biotin D-sulfoxide (BSO). The BBP-A protein binds free biotin with high, "streptavidin-like" affinity (Kd ~ 10-13 M), which is about 50 times lower than that of chicken avidin. Surprisingly, the affinity of BBP-A for BSO is even higher than the affinity for BTN. Furthermore, the solved structures of the BBP-A--BTN and BBP-A--BSO complexes, which share the fold with the members of the avidin and lipocalin protein families, are extremely similar to each other. CONCLUSION: BBP-A is an avidin-like protein having a beta-barrel fold and high affinity towards BTN. However, BBP-A differs from the other known members of the avidin protein family in thermal stability and immunological properties. BBP-A also has a unique ligand-binding property, the ability to bind BTN and BSO at comparable affinities. BBP-A may have use as a novel material in, e.g. modern bio(nano)technological applications.

Binding properties of HABA-type azo derivatives to avidin and avidin-related protein 4.

The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D-biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands beta3 and beta4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray structure for avidin reported here. These results may be valuable in designing improved tools for avidin-based life science and nanobiotechnology applications.

Structural characterization of core-bradavidin in complex with biotin.

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 ("Brad-tag") act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin-Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.

Artificial Avidin-Based Receptors for a Panel of Small Molecules.

Proteins with high specificity, affinity, and stability are needed for biomolecular recognition in a plethora of applications. Antibodies are powerful affinity tools, but they may also suffer from limitations such as low stability and high production costs. Avidin and streptavidin provide a promising scaffold for protein engineering, and due to their ultratight binding to D-biotin they are widely used in various biotechnological and biomedical applications. In this study, we demonstrate that the avidin scaffold is suitable for use as a novel receptor for several biologically active small molecules: Artificial, chicken avidin-based proteins, antidins, were generated using a directed evolution method for progesterone, hydrocortisone, testosterone, cholic acid, ketoprofen, and folic acid, all with micromolar to nanomolar affinity and significantly reduced biotin-binding affinity. We also describe the crystal structure of an antidin, sbAvd-2(I117Y), a steroid-binding avidin, which proves that the avidin scaffold can tolerate significant modifications without losing its characteristic tetrameric beta-barrel structure, helping us to further design avidin-based small molecule receptors.

Crystal structure of the human vascular adhesion protein-1: unique structural features with functional implications.

The expression of human vascular adhesion protein-1 (hVAP-1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X-ray structure of hVAP-1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane-bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP-1, especially regarding the role of hVAP-1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N-glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent-exposed RGD motif at the entrance to each active site in hVAP-1 suggests that it may have a functional role.

Avidin related protein 2 shows unique structural and functional features among the avidin protein family.

BACKGROUND: The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin. RESULTS: In the present study, we have employed multiple biochemical methods to better understand the structure-function relationship of AVR proteins focusing on AVR2. Firstly, we have solved the high-resolution crystal structure of AVR2 in complex with a bound ligand, D-biotin. The AVR2 structure reveals an overall fold similar to the previously determined structures of avidin and AVR4. Major differences are seen, especially at the 1-3 subunit interface, which is stabilized mainly by polar interactions in the case of AVR2 but by hydrophobic interactions in the case of AVR4 and avidin, and in the vicinity of the biotin binding pocket. Secondly, mutagenesis, competitive dissociation analysis and differential scanning calorimetry were used to compare and study the biotin-binding properties as well as the thermal stability of AVRs and avidin. These analyses pinpointed the importance of residue 109 for biotin binding and stability of AVRs. The I109K mutation increased the biotin-binding affinity of AVR2, whereas the K109I mutation decreased the biotin-binding affinity of AVR4. Furthermore, the thermal stability of AVR2(I109K) increased in comparison to the wild-type protein and the K109I mutation led to a decrease in the thermal stability of AVR4. CONCLUSION: Altogether, this study broadens our understanding of the structural features determining the ligand-binding affinities and stability as well as the molecular evolution within the protein family. This novel information can be applied to further develop and improve the tools already widely used in avidin-biotin technology.

Structure-function analysis of enoyl thioester reductase involved in mitochondrial maintenance.

Candida tropicalis enoyl thioester reductase Etr1p and the Saccharomyces cerevisiae homologue Mrf1p catalyse the NADPH-dependent reduction of trans-2-enoyl thioesters in mitochondrial fatty acid synthesis (FAS). Unlike prokaryotic enoyl thioester reductases (ETRs), which belong to the short-chain dehydrogenases/reductases (SDR), Etr1p and Mrf1p represent structurally distinguishable ETRs that belong to the medium-chain dehydrogenases/reductases (MDR) superfamily, indicating independent origin of two separate classes of ETRs. The crystal structures of Etr1p, the Etr1p-NADPH complex and the Etr1Y79Np mutant were refined to 1.70A, 2.25A and 2.60A resolution, respectively. The native fold of Etr1p was maintained in Etr1Y79Np, but the mutant had only 0.1% of Etr1p catalytic activity remaining and failed to rescue the respiratory deficient phenotype of the mrf1Delta strain. Mutagenesis of Tyr73 in Mrf1p, corresponding to Tyr79 in Etr1p, produced similar results. Our data indicate that the mitochondrial reductase activity is indispensable for respiratory function in yeast, emphasizing the significance of Mrf1p (Etr1p) and mitochondrial FAS for the integrity of the respiratory competent organelle.

Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli.

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.

A novel chimeric avidin with increased thermal stability using DNA shuffling.

Avidins are a family of proteins widely employed in biotechnology. We have previously shown that functional chimeric mutant proteins can be created from avidin and avidin-related protein 2 using a methodology combining random mutagenesis by recombination and selection by a tailored biopanning protocol (phage display). Here, we report the crystal structure of one of the previously selected and characterized chimeric avidin forms, A/A2-1. The structure was solved at 1.8 Å resolution and revealed that the protein fold was not affected by the shuffled sequences. The structure also supports the previously observed physicochemical properties of the mutant. Furthermore, we improved the selection and screening methodology to select for chimeric avidins with slower dissociation rate from biotin than were selected earlier. This resulted in the chimeric mutant A/A2-B, which showed increased thermal stability as compared to A/A2-1 and the parental proteins. The increased stability was especially evident at conditions of extreme pH as characterized using differential scanning calorimetry. In addition, amino acid sequence and structural comparison of the chimeric mutants and the parental proteins led to the rational design of A/A2-B I109K. This mutation further decreased the dissociation rate from biotin and yielded an increase in the thermal stability.

Zebavidin--an avidin-like protein from zebrafish.

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.