Heterologous production and characterization of selected secondary active transporters from the CDF, KUP, MOP, FNT, RhtB and SulP families

Genes coding for membrane proteins make up 25%-30% of the genome in most organisms. Membrane proteins play an important role in cell functioning and their importance is enhanced by the fact that a large number of drugs are targeted at membrane proteins. Paradoxically, experimentally determined structures of membrane protein correspond to only about 1.7% of protein structures deposited in the protein data bank (PDB). This is largely due to the fact that membrane proteins are difficult to deal with owing to their amphipathic nature. The low abundance of membrane proteins in native tissue makes heterologous overexpression of these genes a necessity. This thesis work aimed at heterologous production of several secondary active transporter proteins for structural and functional characterizations and establishing alternative strategies to overcome the obstacles associated with heterologous overproduction. Four members of the heavy metal transporting cation diffusion facilitator (CDF) family from S. typhimurium and A. aeolicus were heterologously overproduced in E. coli and functionally characterized by an in vivo complementation assay using the zinc transport deficient E. coli GG48 strain. Out of these four, Aq_2073 from A. aeolicus was produced in large scale with substantial yield and purity sufficient to carry out structural studies. After extensive stability studies with different detergents, pHs and temperatures, the protein was subjected to 3D and 2D crystallization trials. Several C- terminal truncated constructs were made and the simultaneous crystallization screenings were carried out. These resulted in initial needle like crystals in 3D crystallization trials or optimum sized vesicles with crystalline patches in 2D crystallization trials but no obvious crystal. The protein showed significant increase in melting temperature in the presence of cadmium, when tested by differential scanning calorimetry. Another transporter, STM3880 of the potassium uptake permease (KUP) family from S. typhimurium, was heterologously overproduced in E. coli, purified by affinity chromatography, reconstituted into artificial liposome and functionally characterized by solid supported membrane based electrophysiology. In order to establish alternative expression strategies, continuous exchange cell free expression (CECF) of proteins from four different families was carried out. This method found to be aptly complementing the cell-based production approach. Targets from resistance to homoserine/threonine (RhtB) family not expressing in vivo could be expressed and purified using CECF. STM1781 of the sulfate permease (SulP) family was expressed, purified and characterized for stability while the cell-based production resulted in extensive degradation. PF0780 of multidrug/oligosaccharidyllipid/polysaccharide flippase (MOP) family was also purified to homogeneity and the stability was comparable to in vivo produced protein. Moreover, the effect of maltose binding protein (MBP) fusion at N-terminus on production and membrane integration was tested with three selected targets. The analysis revealed decreased yields in the presence of MBP if the protein had both termini in the cytoplasm. This work succeed in heterologously overproducing and establishing purification protocols for several secondary active transporters aiming at structural and functional characterization in a structural genomics framework. It also showed that integration of alternative strategies, like employing both cell-based and cell-free heterologous expression systems, expands the overall expression space coverage and in turn increases the chance of success of a structural genomics styled project.In den meisten Organismen kodieren 25%-30% des Genoms für Membranproteine. Sie spielen eine wichtige Rolle für die Zellfunktion, und ihre Bedeutung wird verstärkt durch die Tatsache, dass viele Medikamente an Membranproteinen angreifen. Paradoxerweise entfallen lediglich 1,7% der hinterlegten Proteinstrukturen in der protein databank (PDB) auf experimentell ermittelte Strukturen von Membranproteinen. Dies ist hauptsächlich auf die schwierige Handhabung, bedingt durch ihren amphipathischen Charakter, zurückzuführen. Das geringe Vorkommen in natürlichen Geweben macht die heterologe Überexpression dieser Gene zu einer Notwendigkeit. Diese Arbeit zielte darauf ab, verschiedene sekundär aktive Transportproteine für strukturelle und funktionelle Untersuchungen heterolog zu produzieren und alternative Strategien zu etablieren, um die Hürden im Zusammenhang mit der heterologen Überexpression zu überwinden. Vier Mitglieder der Schwermetall-transportierenden cation diffusion facilitator (CDF) Familie aus S. typhimurium und A. aeolicus wurden heterolog in E. coli produziert und durch einen in vivo Komplementationsassay unter Zuhilfenahme des Zink-Transport defizienten E. coli Stammes GG48 funktionell charakterisiert. Von diesen vier Proteinen konnte Aq_2073 aus A. aeolicus im präparativen Maßstab mit einer beachtlichen Ausbeute und Reinheit hergestellt werden, um strukturelle Untersuchungen durchzuführen. Nach umfangreichen Stabilitätsuntersuchungen mit verschiedenen Detergenzien, pH-Werten und Temperaturen wurden 2D sowie 3D-Kristallisationsversuche unternommen. Zusätzlich wurden mehrere C-terminal verkürzte Konstrukte hergestellt, welche ebenfalls für Kristallisationsversuche genutzt wurden. Diese ergaben in 3D-Kristallisationsversuchen erste nadelähnliche Kristalle oder in 2D-Kristallisationsversuchen Vesikel mit kristallinen Stellen, aber keine offensichtlichen Kristalle. Das Protein besaß eine signifikant höhere Schmelztemperatur in Gegenwart von Cadmiumionen wie durch differential scanning calorimetry (DSC) gezeigt wurde. Ein weiterer Transporter, STM3880 aus der potassium uptake permease (KUP) Familie aus S. typhimurium, wurde in E. coli heterolog exprimiert, durch Affinitätschromatographie gereinigt, in künstliche Liposomen rekonstituiert und durch elektrophysiologische Untersuchungen mit Festphasen-gestützten Membranen (solid supported membranes; SSM) funktionell charakterisiert. Um alternative Expressionsstrategien zu etablieren, wurden Proteine von vier verschiedenen Familien zellfrei unter kontinuierlichem Austausch (continuous exchange cell free expression; CECF) produziert. Diese Methode schien geeignet, um den zellbasierten Ansatz zu komplementieren. Zielproteine der resistence to homoserine/threonine (RhtB) Familie, die in vivo keine Expression zeigten, konnten so hergestellt und gereinigt werden. STM1781 aus der sulfate permease (SulP) Familie konnte zellfrei hergestellt, gereinigt und hinsichtlich seiner Stabilität charakterisiert werden, wohingegen die zellbasierte Produktion in starkem Abbau resultierte. PF0780 der multidrug/oligosaccharidyl-lipid/polysaccharide flippase (MOP) Familie wurde ebenfalls zur Homogenität gereinigt und zeigte eine vergleichbare Stabilität zum in vivo produzierten Protein. Des Weiteren wurde der Effekt einer Fusion des Maltose bindenden Proteins (MBP) am N-Terminus auf die Produktion und Membranintegration bei drei verschiedenen Proteinen untersucht. Die Analyse ergab eine verringerte Ausbeute in Anwesenheit des MBPs, wenn sich beide Termini des Proteins im Cytoplasma befanden. In dieser Arbeit konnten verschiedene sekundär aktive Transporter erfolgreich hergestellt und entsprechende Reinigungsprotokolle etabliert werden, die darauf abzielen strukturelle und funktionelle Untersuchungen im Rahmen eines structural genomics Projektes durchzuführen. Es zeigte sich, dass die Einbindung alternativer Strategien wie die Anwendung zellbasierter und zellfreier Expressionssysteme, die Gesamtanzahl herstellbarer Proteine steigert und damit die Erfolgsaussichten derart angelegter Projekte verbessert

Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.

The glucocorticoid receptor (GR), like many signaling proteins, depends on the Hsp90 molecular chaperone for in vivo function. Although Hsp90 is required for ligand binding in vivo, purified apo GR is capable of binding ligand with no enhancement from Hsp90. We reveal that Hsp70, known to facilitate client delivery to Hsp90, inactivates GR through partial unfolding, whereas Hsp90 reverses this inactivation. Full recovery of ligand binding requires ATP hydrolysis on Hsp90 and the Hop and p23 cochaperones. Surprisingly, Hsp90 ATP hydrolysis appears to regulate client transfer from Hsp70, likely through a coupling of the two chaperone's ATP cycles. Such coupling is embodied in contacts between Hsp90 and Hsp70 in the GR:Hsp70:Hsp90:Hop complex imaged by cryoelectron microscopy. Whereas GR released from Hsp70 is aggregation prone, release from Hsp90 protects GR from aggregation and enhances its ligand affinity. Together, this illustrates how coordinated chaperone interactions can enhance stability, function, and regulation

Structural Correlates of the Temperature Sensitive Phenotype Derived from Saturation Mutagenesis Studies of CcdB

Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4

Histone H3 binding to the PHD1 domain of histone demethylase KDM5A enables active site remodeling.

Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation

Differential Isotopic Enrichment To Facilitate Characterization of Asymmetric Multimeric Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein–ligand and protein–protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched (¹⁵N-labeled) p51 and unlabeled p66. To enable detection of ¹⁵N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% ¹⁵N model. Our results demonstrated that ¹⁵N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but ¹⁵N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure

Identification of a small molecular insulin receptor agonist with potent antidiabetes activity

Insulin replacement therapy is a widely adopted treatment for all patients with type 1 diabetes and some with type 2 diabetes. However, injection of insulin has suffered from problems such as tissue irritation, abscesses, discomfort, and inconvenience. The use of orally bioactive insulin mimetics thus represents an ideal treatment alternative. Here we show that a chaetochromin derivative (4548-G05) acts as a new nonpeptidyl insulin mimetic. 4548-G05 selectively activates an insulin receptor (IR) but not insulin-like growth factor receptor-I or other receptor tyrosine kinases. Through binding to the extracellular domain of the IR, 4548-G05 induces activation of the receptor and initiates the downstream Akt and extracellular signal-related kinase pathways to trigger glucose uptake in C2C12 myotubes. Moreover, it displays a potent blood glucose-lowering effect when administrated orally in normal, type 1 diabetic, and type 2 diabetic mice models. Therefore, 4548-G05 may represent a novel pharmacological agent for antidiabetes drug development. © 2014 by the American Diabetes Association.Link_to_subscribed_fulltex

Crystal structures of the nuclear receptor, liver receptor homolog 1, bound to synthetic agonists

Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. While preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid (PL) LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth, nor do they expand the pocket like PLs. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable, face-to-face ?-?-stacking with LRH-1 residue H390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies

Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein–ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test–retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error