Strukturen und Ligandenerkennung eines WW-Domaenenpaares und einer FF-Domaene

Title and Contents 1  Introduction                                                                                                1 2  NMR spectroscopy                                                                                  13 3  Material and Methods                                                                              29 4  Binding of Pro-rich ligands to WW and SH3 domains                         52 5  Solution structure of the Prp40 WW domain pair                                  66 6  Ligand recognition of the Prp40 WW domain pair                                81 7  Structure and interactions of the Prp40 FF1 domain                            96     Abbreviations, symbols and units                                                          113     Appendix A: Experimental data                                                             119Many eukaryotic proteins encompass multiple cooperating domains and/or domain repeats fine- tuning the functions of these so-called mosaic proteins. It is therefore of key interest to know not only the structures of the individual domains constituting a protein, but also their relative arrangement. The splicing factor Prp40 (pre-mRNA processing protein) comprises an N-terminal WW domain pair and six consecutive FF domains. In the yeast spliceosome, Prp40 is associated with the U1 snRNP (small nuclear ribonucleoprotein) and interacts with a number of splicing factors functioning in cross-intron bridging. In this Thesis, the solution structures of the tandem WW domains and the N-terminal FF domain of Prp40 have been determined by NMR (nuclear magnetic resonance) spectroscopy. To study the interaction of these Prp40 domains with the above-mentioned spliceosomal binding partners, 1H- 15N correlation spectra were recorded to map changes in the resonance frequencies of the domains upon ligand binding. It was found that the WW domains fold as triple-stranded anti- parallel beta- sheets, which are not in direct contact, but separated by a stable alpha-helical linker. Using 1H-155N residual dipolar couplings (RDCs) the relative orientation of the WW domains has been precisely defined despite the lack of inter-domain distance restraints, revealing that the binding pockets face opposite sides. This supports the idea that the two Prp40 WW domains function as central adaptors in early spliceosomal complexes, bringing two different binding partners together. This suggestion has been confirmed by a detailed analysis of the proline-rich motifs recognised by the Prp40 WW domains. Both Prp40 WW domains are shown to interact with PPxY motifs present in the splicing factors BBP and Prp8, but also with related peptides devoid of aromatic residues. However, no interaction was observed between the Prp40 WW domains and the CTD repeats used questioning the direct function suggested for the Prp40 WW domains in coordinating splicing and transcription. The N-terminal FF domain of Prp40 exhibits a novel alpha-helical fold. The three-dimensional structure of the Prp40 FF1 domain consists of four tightly packed alpha-helices. The second alpha- helix and its surrounding residues are shown to constitute the binding surface, which can accommodate the N-terminal TPR motif of the spliceosomal protein Clf1. These findings further cement the role of Prp40 as a central adaptor unit in the early spliceosome. Given that lethal U1 snRNA mutations can be suppressed by a point mutation in the second Prp40 FF domain (FF2), a homology model of the FF2 domain has been generated based on the determined FF1 domain structure. This model reveals significant differences in the electrostatic surface potential of the FF domain binding sites. In contrast to the FF1 domain, the binding surface of the FF2 domain is highly positively charged suggesting that the Prp40 FF2 domain can directly bind the U1 snRNA, although its sequence does not display any known RNA recognition motif. In summary, the data presented in this Thesis shed new light onto the structural arrangement of splicing factors present in the yeast pre- spliceosome. The described results suggest the formation of a tight complex between the U1 snRNP, Prp40, BBP, Prp8 and Clf1 in the early spliceosome, which brings the 5' and the 3' splice-site into spatial proximity.Viele eukaryotische Proteine bestehen aus Domänen, deren Zusammenspiel die Funktion sogenannter Mosaikproteine oft entscheidend beeinflußt. Es ist daher von besonderem Interesse, nicht nur die Strukturen einzelner Proteindomänen zu kennen, sondern auch deren relative Anordnung. Der Spleißfaktor Prp40 (pre- mRNA processing protein) ist ein Mosaikprotein, das aus einem N-terminalen WW- Domänenpaar und sechs darauffolgenden FF-Domänen besteht. Im Hefe-Spleißosom bringt Prp40 die 3'-und 5'-Spleißstelle in räumliche Nähe. In dieser Arbeit wurden die Strukturen des WW-Domänenpaares und der N-terminalen FF- Domäne des Spleißfaktors Prp40 mittels NMR-Spektroskopie (Kernspinresonanz oder nuclear magnetic resonance) bestimmt. Mit Hilfe von 1H-15N Korrelationsspektren wurde außerdem die Wechselwirkung dieser Prp40-Domänen mit den obengenannten Spleißfaktoren anhand der Resonanzfrequenzänderungen der Domänen bei Zugabe von Liganden studiert. Es konnte gezeigt werden, daß die WW-Domänen sich zu drei-strängigen anti-parallelen beta-Faltblättern falten, die durch einen stabilen alpha-helikalen Linker räumlich voneinander getrennt sind. Die relative Domänenanordnung zeigt, daß die Bindungstaschen der WW-Domänen in entgegengesetzte Richtungen zeigen. Dieses Ergebnis bestätigt die Vermutung, daß die Prp40 WW-Domänen als zentrale Adapter im frühen Hefe-Spleißosom fungieren und zwei verschiedene Bindungspartner in räumliche Nähe bringen können. Weitere Hinweise, die diese Vermutung stützen, erhielt man aus einer detaillierten Analyse Prolin-reicher Motive, die an die WW-Domänen des Prp40-Proteins binden. Diese Analyse zeigte, daß beide WW-Domänen an die PPxY- Motive der Spleißfaktoren BBP und Prp8 binden. Außerdem wurden Wechselwirkungen zwischen den Prp40 WW-Domänen und verwandten Prolin-reichen Peptiden beobachtet, denen ein aromatischer Rest fehlte. Im Gegensatz dazu fand keine Wechselwirkung zwischen den Prp40 WW-Domänen und den verwendeten RNA Polymerase II CTD-Sequenzen statt, was an einer direkten Funktion der Prp40 WW-Domänen bei der Koordination von Transkription und Spleißen zweifeln läßt. Die N-terminale FF(FF1)-Domäne des Prp40-Proteins weist eine neuartige alpha-helikale Struktur auf. Die dreidimensionale Struktur besteht aus vier dicht gepackten alpha-Helices. Die zweite alpha- Helix bildet zusammen mit den umgebenden Seitenketten die Bindungsfläche für das N-terminale TPR-Motiv des Spleißfaktors Clf1. Diese Ergebnisse untermauern die Rolle von Prp40 als zentraler Bindungsplattform im frühen Spleißosom. Da bekannt ist, daß eine Punktmutation in der zweiten Prp40 FF-Domäne (FF2) letale U1 snRNA-Mutationen unterdrückt, wurde ausgehend von der bereits bestimmten Struktur der FF1-Domäne ein Homologiemodell der FF2-Domäne erstellt. Dieses Modell weist signifikante Unterschiede im elektrostatischen Oberflächenpotential der Bindungstellen dieser beiden FF-Domänen auf. Im Gegensatz zur FF1-Domäne ist die Bindungstelle der FF2-Domäne stark positiv geladen. Dies läßt vermuten, daß die FF2-Domäne direkt an U1 snRNA binden kann, obwohl ihre Aminosäuresequenz kein bisher bekanntes RNA-Erkennungsmotiv aufweist. Abschließend kann man sagen, daß diese Arbeit neue Einblicke in die strukturelle Anordnung von Spleißfaktoren im frühen Hefe-Spleißosom liefert. Anhand der hier präsentierten Ergebnisse läßt sich ein Modell vorschlagen, in dem das U1 snRNP mit den Spleißfaktoren Prp40, BBP, Prp8 und Clf1 ein dichtes Protein-RNA-Netzwerk bildet, das dazu dient, die 3'- und 5'-Spleißstelle in räumliche Nähe zu bringen

Methionine Scanning as an NMR Tool for Detecting and Analyzing Biomolecular Interaction Surfaces

SummaryMethyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments

β-Sheet Augmentation Is a Conserved Mechanism of Priming HECT E3 Ligases for Ubiquitin Ligation

Ubiquitin (Ub) ligases (E3s) catalyze the attachment of Ub chains to target proteins and thereby regulate a wide array of signal transduction pathways in eukaryotes. In HECT-type E3s, Ub first forms a thioester intermediate with a strictly conserved Cys in the C-lobe of the HECT domain and is then ligated via an isopeptide bond to a Lys residue in the substrate or a preceding Ub in a poly-Ub chain. To date, many key aspects of HECT-mediated Ub transfer have remained elusive. Here, we provide structural and functional insights into the catalytic mechanism of the HECT-type ligase Huwe1 and compare it to the unrelated, K63-specific Smurf2 E3, a member of the Nedd4 family. We found that the Huwe1 HECT domain, in contrast to Nedd4-family E3s, prioritizes K6- and K48-poly-Ub chains and does not interact with Ub in a non-covalent manner. Despite these mechanistic differences, we demonstrate that the architecture of the C-lobe Ub intermediate is conserved between Huwe1 and Smurf2 and involves a reorientation of the very C-terminal residues. Moreover, in Nedd4 E3s and Huwe1, the individual sequence composition of the Huwe1 C-terminal tail modulates ubiquitination activity, without affecting thioester formation. In sum, our data suggest that catalysis of HECT ligases hold common features, such as the 13-sheet augmentation that primes the enzymes for ligation, and variable elements, such as the sequence of the HECT C-terminal tail, that fine-tune ubiquitination activity and may aid in determining Ub chain specificity by positioning the substrate or acceptor Ub. (C) 2018 Elsevier Ltd. All rights reserved

The WW1 Domain Enhances Autoinhibition in Smurf Ubiquitin Ligases

Downregulation of ubiquitin (Ub) ligase activity prevents premature ubiquitination and is critical for cellular homeostasis. Nedd4 Ub ligases share a common domain architecture and yet are regulated in distinct ways through interactions of the catalytic HECT domain with the N-terminal C2 domain or the central WW domain region. Smurf1 and Smurf2 are two highly related Nedd4 ligases with similar to 70% overall sequence identity. Here, we show that the Smurf1 C2 domain interacts with the HECT domain and inhibits ligase activity in trans. However, in contrast to Smurf2, we find that full-length Smurf1 is a highly active Ub ligase, and we can attribute this striking difference in regulation to the lack of one WW domain (WW1) in Smurf1. Using NMR spectroscopy and biochemical assays, we identified the WW1 region as an additional inhibitory element in Smurf2 that cooperates with the C2 domain to enhance HECT domain binding and Smurf2 inhibition. Our work provides important insights into Smurf regulation and highlights that the activities of highly related proteins can be controlled in distinct ways. (C) 2019 Elsevier Ltd. All rights reserved