Force and affinity in cellulosomal complexes

In dieser Arbeit werden die molekularen Mechanismen der Organisation des Cellulosoms - ein komplexes extrazelluläres Proteinnetzwerk - als Modellsystem für Protein-Protein Interaktionen mittels biophysikalischer Methoden untersucht. Dieses extrazelluläre Organell ermöglicht bestimmten Bakterien die Zersetzung von Cellulose, indem es Enzyme und Cellulose-Bindedomänen auf gerüstartigen Proteinstrukturen in synergistischer Weise kombiniert. Die einzelnen Komponenten werden hierbei von einer Klasse von Rezeptor-Liganden-Paaren namens Cohesin- Dockerin in ihrer Stöchiometrie und Anordnung funktionell kombiniert. Ein Teil dieser Arbeit besteht in der Entschlüsselung der molekularen Bindemechanismen des Cohesins CohE, welches das Bakterium Ruminococcus flavefaciens mit seinem Cellusom verbindet. Durch die Kombination von Einzelmolekül-Kraftspektroskopie mit Molekulardynamik-Simulationen konnte die aussergewöhnliche Belastbarkeit der Interaktionen von CohE mit zwei homologen Dockerinen entschlüsselt werden. Hierbei wurde insbesondere der Einfluss der Kraftpropagation innerhalb eines Proteinkomplexes auf dessen mechanische Widerstandsfähigkeit untersucht. Die physiologische Verankerung über den carboxyl-Terminus von CohE erwies sich als deutlich robuster im Vergleich zu einer nicht nativen N-terminalen Verankerung. Um den Kontrast zwischen hoher mechanischer Belastbarkeit bei moderaten Affinitäten im nano- bis mikromolaren Bereich besser verstehen zu können, wandte ich mich der Bestimmung der kinetischen Ratenkonstanten koff und kon zu, deren Quotient die Gleichgewichtskonstante bildet. Während es eine kleine Dissoziationskonstante dem Bakterium ermöglichen würde die von ihm exprimierte Nanomaschinerie fest an sich zu binden, könnte ein höheres koff und kon einen dynamischeren Austausch von Cellulosomen innerhalb des Mikrobioms ermöglichen. Zusätzlich stellte sich die Frage, ob die Verankerungsgeometrie auch in Abwesenheit von Kraft Einfluss auf das Bindeverhalten nehmen würde. Nachdem initiale Messungen mittels Oberflächenplasmonenresonanzspektroskopie inkonsistent waren, wurde eine neuartige, enzymbasierte Kopplungsstrategie für oberflächengebundene Affinitätsbestimmungen entwickelt. Hiermit konnte CohE funktional und spezifisch auf Sensoroberflächen immobilisiert werden. Es zeigte sich, dass in Abwesenheit von externer Kraft die Verankerungsgeometrie von CohE keinen Einfluss auf das Bindeverhalten hat. Dies bestärkt im Umkehrschluss die Hypothese, dass mechanische Stabilitäten stets geometrieabhängig zu untersuchen sind. Im Rahmen dieser Arbeit wurden auch methodische Verbesserungen in der Einzelmolekülkraftspektroskopie erzielt. Zum einen wurde eine Strategie entwickelt, um Proteindomänen zeitsparend in vitro zu exprimieren und ohne weitere Aufreinigung spezifisch auf Objektträgern zu verankern. Die darauffolgende enzymatische Peptidligation eines Dockerins via Sortase A erlaubt es nun, mit hohem Durchsatz Entfaltungsstudien an Proteinen mithilfe der Cohesin-Dockerin Interaktion durchzuführen. Weiterhin ermöglichte es dieselbe Sortase-vermittelte Peptidligation, die gängigen Polyethylenlinker durch Elastin-ähnliche Peptide zu ersetzen. Dies verhindert Artefakte, die sonst durch Polyethylenlinker bei Protein-Kraftspektroskopie über 100 pN entstünden. Zuletzt wurde der Entfaltungsprozess einer Cohesin-Domäne aus Acetivibrio cellulolyticus untersucht, deren Familie in vorangegangenen Studien teils bimodale Entfaltungskraftverteilungen zeigte. Durch die Kombination zweier Messmodi konnte die Kraft-Ladungsrate über fünf Größenordnungen variiert werden. Es konnte gezeigt werden, dass das dabei beobachtete Verhalten mit einer Konformationsänderung während der experimentellen Zeitskala zwischen verschiedenen, gefalteten Konformationen konsistent ist

Mechanisms of Nanonewton Mechanostability in a Protein Complex Revealed by Molecular Dynamics Simulations and Single-Molecule Force Spectroscopy

Can molecular dynamics simulations predict the mechanical behavior of protein complexes? Can simulations decipher the role of protein domains of unknown function in large macromolecular complexes? Here, we employ a wide-sampling computational approach to demonstrate that molecular dynamics simulations, when carefully performed and combined with single-molecule atomic force spectroscopy experiments, can predict and explain the behavior of highly mechanostable protein complexes. As a test case, we studied a previously unreported homologue from; Ruminococcus flavefaciens; called X-module-Dockerin (XDoc) bound to its partner Cohesin (Coh). By performing dozens of short simulation replicas near the rupture event, and analyzing dynamic network fluctuations, we were able to generate large simulation statistics and directly compare them with experiments to uncover the mechanisms involved in mechanical stabilization. Our single-molecule force spectroscopy experiments show that the XDoc-Coh homologue complex withstands forces up to 1 nN at loading rates of 10; 5; pN/s. Our simulation results reveal that this remarkable mechanical stability is achieved by a protein architecture that directs molecular deformation along paths that run perpendicular to the pulling axis. The X-module was found to play a crucial role in shielding the adjacent protein complex from mechanical rupture. These mechanisms of protein mechanical stabilization have potential applications in biotechnology for the development of systems exhibiting shear enhanced adhesion or tunable mechanics

Ultrastable cellulosome-adhesion complex tightens under load

Challenging environments have guided nature in the development of ultrastable protein complexes. Specialized bacteria produce discrete multi-component protein networks called cellulosomes to effectively digest lignocellulosic biomass. While network assembly is enabled by protein interactions with commonplace affinities, we show that certain cellulosomal ligand-receptor interactions exhibit extreme resistance to applied force. Here, we characterize the ligand-receptor complex responsible for substrate anchoring in the Ruminococcus flavefaciens cellulosome using single-molecule force spectroscopy and steered molecular dynamics simulations. The complex withstands forces of 600-750 pN, making it one of the strongest bimolecular interactions reported, equivalent to half the mechanical strength of a covalent bond. Our findings demonstrate force activation and inter-domain stabilization of the complex, and suggest that certain network components serve as mechanical effectors for maintaining network integrity. This detailed understanding of cellulosomal network components may help in the development of biocatalysts for production of fuels and chemicals from renewable plant-derived biomass

Elastin-like Polypeptide Linkers for Single-Molecule Force Spectroscopy

Single-molecule force spectroscopy (SMFS) is by now well established as a standard technique in biophysics and mechanobiology. In recent years, the technique has benefitted greatly from new approaches to bioconjugation of proteins to surfaces. Indeed, optimized immobilization strategies for biomolecules and refined purification schemes are being steadily adapted and improved, which in turn has enhanced data quality. In many previously reported SMFS studies, poly(ethylene glycol) (PEG) was used to anchor molecules of interest to surfaces and/or cantilever tips. The limitation, however, is that PEG exhibits a well-known trans-trans-gauche to all-trans transition, which results in marked deviation from standard polymer elasticity models such as the worm-like chain, particularly at elevated forces. As a result, the assignment of unfolding events to protein domains based on their corresponding amino acid chain lengths is significantly obscured. Here, we provide a solution to this problem by implementing unstructured elastin-like polypeptides as linkers to replace PEG. We investigate the suitability of tailored elastin-like polypeptides linkers and perform direct comparisons to PEG, focusing on attributes that are critical for single-molecule force experiments such as linker length, monodispersity, and bioorthogonal conjugation tags. Our results demonstrate that by avoiding the ambiguous elastic response of mixed PEG/peptide systems and instead building the molecular mechanical systems with only a single bond type with uniform elastic properties, we improve data quality and facilitate data analysis and interpretation in force spectroscopy experiments. The use of all-peptide linkers allows alternative approaches for precisely defining elastic properties of proteins linked to surfaces

Improved ANAP incorporation and VCF analysis reveal details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain

The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing “cap” domain, and a globular “ballast domain” containing GTP/GDP and dinuclear Zn2+-binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)–2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry. While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only 3 out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented

Post-Translational Sortase-Mediated Attachment of High-Strength Force Spectroscopy Handles

Single-molecule force spectroscopy greatly benefits from site-specific surface immobilization and specific probing with a functionalized cantilever. Here, we describe a streamlined approach to such experiments by covalently attaching mechanically stable receptors onto proteins of interest (POI) to improve pickup efficiency and specificity. This platform provides improved throughput, allows precise control over the pulling geometry, and allows for multiple constructs to be probed with the same ligand-modified cantilever. We employ two orthogonal enzymatic ligation reactions [sortase and phosphopantetheinyl transferase (Sfp)] to covalently immobilize POI to a pegylated surface and to subsequently ligate the POI to a mechanically stable dockerin domain at the protein’s C-terminus for use as a high-strength pulling handle. Our configuration permits expression and folding of the POI to proceed independently from the mechanically stable receptor used for specific probing and requires only two short terminal peptide sequences (i.e., ybbR-tag and sortase C-tag). We applied this system successfully to proteins expressed using in vitro transcription and translation reactions without a protein purification step and to purified proteins expressed in Escherichia coli

Structural and mechanistic insights into mechanoactivation of focal adhesion kinase

9 p.-7 fig.-1 tab.Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.C.D. and F.G. are grateful for support from the state of Baden-Wu¨ rttemberg through high performance computing in Baden-Wu¨ rttemberg (bwHPC) and Deutsche Forschungsgemeinschaft (DFG) Grant INST 35/1134-1 FUGG. H.E.G. acknowledges funding from DFG Grant Sonderforschungsbereich 1032. F.G. acknowledges funding from the DFG through the research group SHENC (Shear Flow Regulation of Hemostasis—Bridging the Gap Between Nanomechanics and Clinical Presentation) and from the Klaus Tschira Foundation. D.L. acknowledges support from Spanish Ministry of Economy, Industry and Competitiveness Retos Grant BFU2016-77665-R cofunded by the European Regional Development Fund and Volkswagen Foundation Grant Az: 86 416-1. D.L. is the recipient of Worldwide Cancer Research Award 15-1177.Peer reviewe

Mapping Mechanical Force Propagation through Biomolecular Complexes

Here we employ single-molecule force spectroscopy with an atomic force microscope (AFM) and steered molecular dynamics (SMD) simulations to reveal force propagation pathways through a mechanically ultrastable multidomain cellulosome protein complex. We demonstrate a new combination of network-based correlation analysis supported by AFM directional pulling experiments, which allowed us to visualize stiff paths through the protein complex along which force is transmitted. The results implicate specific force-propagation routes nonparallel to the pulling axis that are advantageous for achieving high dissociation forces