Kristallographische Studie zum Sexualhormon-bindenden Globulin (SHBG)

Table of contents   Titelblatt List of abbreviations  2 Table of contents 4 1. Scientific background 6 1.1. Introduction 6 1.2. Sex hormone-binding globulin 7 1.3. The aim of my thesis  13 1.4. Short theory of the isomorphous replacement and molecular replacement methods 14 2.  Materials and methods 20 2.1. Preparation of competent E.coli cells 20 2.2. Transformation 20 2.3. Protein production 21 2.4. Protein purification 21 2.5. Polyacrylamide gel electrophoresis 23 2.6. Crystallization  23 2.7. Ultracentrifugation experiments  25 2.8. X-ray diffraction experiments  25 2.9. Structure determination  30 3.  Results 33 3.1. Crystal structure of the N-terminal domain of human SHBG in complex with DHT 33 3.2. The crystal structure of SHBG including the disordered loop region 46 3.3. Structural determinants of the steroid-binding specificity of SHBG 52 4. Discussion 65 4.1. LG domain fold  65 4.2. Dimerization and steroid binding 67 4.3. Zinc effect 69 5. Summary 72 6. Zusammenfassung 74 7. References 76 Acknowledgements 86 Lebenslauf 87 List of Publications  88Abstract: The crystal structure of the amino-terminal LG domain of SHBG in complex with 5a-dihydrotestosterone at 1.55 Å resolution reveals the structure of the LG domain as such as well as the architecture of the steroid-binding site and the quaternary structure of the dimer. The steroid and a 20 Å distant calcium ion are not located at the dimer interface. Instead, two separate steroid binding pockets and calcium binding sites exist per dimer. The crystal structures of the tetragonal crystal form and the EDTA-soaked trigonal crystals of SHBG completed the original structure and reveal the loop segment that covers the steroid-binding pocket. Thus, a more detailed description of the steroid- binding site topography is obtained. The binding of zinc reorients the side- chain of His136 as observed in the original crystal structure of SHBG and in the zinc- complexed crystal structure. Apparently, this residue causes disorder within the loop structure between Pro130 and Arg135. The crystal structures of SHBG in complex with different ligands, which are estradiol, 5a-androstane, 3b,17b-diol (17b-DHA), 5a-androstane, 3b,17a-diol (17a-DHA), 2-methoxyestradiol (methoxyestradiol), norgestrel, have also been solved. Different SHBG-steroid complexes revealed that the steroid-binding pocket of SHBG is very adaptable and displays different possibilities to accommodate the ligands. The binding may occur in two ways, in a "forward" mode like DHT and other chemically related androgens or in the "reverse" mode like in estradiol and it metabolite methoxyestradiol. Depending on which ligand is bound, conformational rearrangements of the residues lining the pocket occur. The X-ray structures of SHBG-steroid complexes presented here provide a three-dimensional composite of key molecular features for the binding of androgens and estrogens.Zusammenfassung: Humanes Sexualhormon-bindendes Globulin (SHBG) transportiert Steroide im Blut und reguliert deren Abgabe an das Gewebe (mit Ausnahme von Progesterone). In biologischen Flüssigkeiten liegt SHBG als Homodimer vor, dessen Monomere sich aus zwei Laminin-G-ähnlichen Domänen (LG-Domänen) zusammensetzten. Die Kristallstruktur der N-terminalen LG-Domäne von SHBG im Komplex mit 5a- Dihydrotestosteron wurde mit einer Auflösung von 1.55 Å gelöst. Die Analyse zeigt die Struktur einer LG-Domäne, die Architektur der Steroid- Bindungsstasche sowie die Struktur der Dimere. Weder das Steroid noch ein Calcium-Ion, das 20 Å entfernt von der Steroid-Bindungsstelle ist, sind in der Dimerisierungskontaktstelle lokalisiert. Stattdessen weist jedes Dimer zwei von einander getrennte Steroid-Bindungstaschen und zwei Calciumbindungsstellen auf. Die Strukturen der tetragonalen Kristalle und der EDTA-getränkten trigonalen Kristalle von SHBG ergänzen die Originalstruktur und zeigen zusätzlich die Schleife, die über der Steroid-Bindungsstelle liegt. Somit ist eine detaillierte Beschreibung der Steroid-Bindungsstelle möglich.Die Bindung von Zink-Ionen führt zu einer Umorientierung der Seitenkette His136, wie sie auch in der Originalstruktur zu sehen ist. Wahrscheinlich gibt es einen Zusammenhang zwischen der Orientierung dieses Restes und der ungeordneten Struktur der Schleife Pro130 bis Arg135. Die Kristallstrukturen von SHBG im Komplex mit Östradiol, 5a-Androstan, 3b ,17b-diol (17b-DHA), 5a-Androstan, 3b,17a-diol (17a-DHA), 2-Methoxyestradiol (Methoxyöstradiol) und Norgestrel konnten ebenfalls bestimmt werden. In den unterschiedlichen SHBG-Komplexstrukturen, dass die Steroid-Bindungstasche sehr flexibel ist und mehrere Möglichkeiten besitzt Liganden zu binden. Nach den hier untersuchten Strukturen zu urteilen, werden die Steroide entweder im "Vorwärts"- Modus wie das DHT und andere chemisch verwandte Androgene oder im "Umkehr"- Modus wie Östradiol und dessen Metabolit Methoxyöstradiol eingebaut. Abhängig von den Liganden kommt es zur Konformationsänderung der Reste, die in der Bindungstasche lokalisiert sind. Die Kristallstrukturen der hier untersuchten SHBG-Komplexe bieten einen strukturellen Überblick über die molekularen Eigenschaften der Androgen- und Östrogenbindung

Foot-and-mouth disease virus leader proteinase: Structural insights into the mechanism of intermolecular cleavage

Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pr)o (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. in intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells. (C) 2014 the Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).Austrian Science FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Med Univ Vienna, Max F Perutz Labs, A-1030 Vienna, AustriaUniv Vienna, Dept Struct & Computat Biol, Max F Perutz Labs, A-1030 Vienna, AustriaUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-0404420 São Paulo, BrazilAustrian Science Foundation: P20889Austrian Science Foundation: P24038FAPESP: 12/50191-4RCNPq: 471340/2011-1CNPq: 470388/2010-2Web of Scienc

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay

The SPOC domain is a phosphoserine binding module that bridges transcription machinery with co- and post-transcriptional regulators

The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of mA, the most abundant RNA modification. RBM15 positively regulates mA levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes

Investigation of Atomic Level Patterns in Protein—Small Ligand Interactions

BACKGROUND: Shape complementarity and non-covalent interactions are believed to drive protein-ligand interaction. To date protein-protein, protein-DNA, and protein-RNA interactions were systematically investigated, which is in contrast to interactions with small ligands. We investigate the role of covalent and non-covalent bonds in protein-small ligand interactions using a comprehensive dataset of 2,320 complexes. METHODOLOGY AND PRINCIPAL FINDINGS: We show that protein-ligand interactions are governed by different forces for different ligand types, i.e., protein-organic compound interactions are governed by hydrogen bonds, van der Waals contacts, and covalent bonds; protein-metal ion interactions are dominated by electrostatic force and coordination bonds; protein-anion interactions are established with electrostatic force, hydrogen bonds, and van der Waals contacts; and protein-inorganic cluster interactions are driven by coordination bonds. We extracted several frequently occurring atomic-level patterns concerning these interactions. For instance, 73% of investigated covalent bonds were summarized with just three patterns in which bonds are formed between thiol of Cys and carbon or sulfur atoms of ligands, and nitrogen of Lys and carbon of ligands. Similar patterns were found for the coordination bonds. Hydrogen bonds occur in 67% of protein-organic compound complexes and 66% of them are formed between NH- group of protein residues and oxygen atom of ligands. We quantify relative abundance of specific interaction types and discuss their characteristic features. The extracted protein-organic compound patterns are shown to complement and improve a geometric approach for prediction of binding sites. CONCLUSIONS AND SIGNIFICANCE: We show that for a given type (group) of ligands and type of the interaction force, majority of protein-ligand interactions are repetitive and could be summarized with several simple atomic-level patterns. We summarize and analyze 10 frequently occurring interaction patterns that cover 56% of all considered complexes and we show a practical application for the patterns that concerns interactions with organic compounds

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.MK was supported by the Vienna Science and Technology Fund (WWTF) through project COV20-031 (to JZ) and a Cambridge Trust LMB Cambridge Scholarship. Research in the AP lab is supported by the Austrian Science Fund (START Projekt Y 1031-B28, SFB “RNA-Deco” F 80) and EMBO-YIP; research in the JB lab is supported by the European Research Council (ERC- 2015-CoG - 682181). The IMP receives generous institutional funding from Boehringer Ingelheim and the Austrian Research Promotion Agency (Headquarter grant FFG-852936); IMBA is generously supported by the Austrian Academy of Sciences. Work in the LM-A laboratory is supported by grant PID2019- 104176RB-I00/AEI/10.13039/501100011033 of the Spanish Ministry of Science and Innovation, and an institutional grant of the Fundación Ramón Areces.Peer reviewe

Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol

In a crystal structure of the amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG), the biologically active estrogen metabolite, 2-methoxyestradiol (2-MeOE2), binds in the same orientation as estradiol. The high affinity of SHBG for 2-MeOE2 relies primarily on hydrogen bonding between the hydroxyl at C-3 of 2-MeOE2 and Asp65 and an interaction between the methoxy group at C-2 and the amido group of Asn82. Accommodation of the 2-MeOE2 methoxy group causes an outward displacement of residues Ser128-Pro130, which appears to disorder and displace the loop region (Leu131-His136) that covers the steroid-binding site. This could influence the binding kinetics of 2-MeOE2 and/or facilitate ligand-dependent interactions between SHBG and other proteins. Occupancy of a zinc-binding site reduces the affinity of SHBG for 2-MeOE2 and estradiol in the same way. The higher affinity of SHBG for estradiol derivatives with a halogen atom at C-2 is due to either enhanced hydrogen bonding between the hydroxyl at C-3 and Asp65 (2-fluoroestradiol) or accommodation of the functional group at C-2 (2-bromoestradiol), rather than an interaction with Asn82. By contrast, the low affinity of SHBG for 2-hydroxyestradiol can be attributed to intra-molecular hydrogen bonding between the hydroxyls in the aromatic steroid ring A, which generates a steric clash with the amido group of Asn82. Understanding how C-2 derivatives of estradiol interact with SHBG could facilitate the design of biologically active synthetic estrogens