Computational modeling of biological nanopores

Throughout our history, we, humans, have sought to better control and understand our environment. To this end, we have extended our natural senses with a host of sensors-tools that enable us to detect both the very large, such as the merging of two black holes at a distance of 1.3 billion light-years from Earth, and the very small, such as the identification of individual viral particles from a complex mixture. This dissertation is devoted to studying the physical mechanisms that govern a tiny, yet highly versatile sensor: the biological nanopore. Biological nanopores are protein molecules that form nanometer-sized apertures in lipid membranes. When an individual molecule passes through this aperture (i.e., "translocates"), the temporary disturbance of the ionic current caused by its passage reveals valuable information on its identity and properties. Despite this seemingly straightforward sensing principle, the complexity of the interactions between the nanopore and the translocating molecule implies that it is often very challenging to unambiguously link the changes in the ionic current with the precise physical phenomena that cause them. It is here that the computational methods employed in this dissertation have the potential to shine, as they are capable of modeling nearly all aspects of the sensing process with near atomistic precision. Beyond familiarizing the reader with the concepts and state-of-the-art of the nanopore field, the primary goals of this dissertation are fourfold: (1) Develop methodologies for accurate modeling of biological nanopores; (2) Investigate the equilibrium electrostatics of biological nanopores; (3) Elucidate the trapping behavior of a protein inside a biological nanopore; and (4) Mapping the transport properties of a biological nanopore. In the first results chapter of this thesis (Chapter 3), we used 3D equilibrium simulations [...]Comment: PhD thesis, 306 pages. Source code available at https://github.com/willemsk/phdthesis-tex

Advancing our understanding of lipid bilayer interactions: a molecular dynamics study

In recent years, advances in computer architecture and lipid force field parameters have made Molecular Dynamics (MD) a powerful tool for gaining atomistic resolution of biological membranes on timescales that other tools simply cannot explore. With many key biological processes involving membranes occurring on the nanosecond timescale, MD allows us to probe the dynamics and energetics of these interactions in molecular detail. Specifically, we can observe the interactions taking place as a peptide or protein comes into contact with a lipid bilayer, and how this may shape or alter the bilayer either locally (changes in headgroup orientation, lipid fluidity) or in bulk (lipid demixing, membrane curvature). The resolution achieved through atomistic MD can be directly compared with other tools such as NMR and EPR to gain a full perspective of how these biological systems behave over different timescales. As my background is in computational physics, this thesis not only looks into broadening our understanding of various interactions with biological membranes, but also into the development of construction and analytical software to assist in my research and benefit others in the field. One aspect of biological membranes that could vastly benefit from MD simulations is that of antimicrobial peptides (AMPs). These peptides primarily target and destroy microbes by permeabilising the cell membrane through a variety of proposed mechanisms, where each mechanism relies on the AMP to adopt specific conformations upon contact with bacterial membranes. In this thesis, I present an investigation into the interactions between a synthetic AMP and an inhibitor peptide designed to regulate antimicrobial activity through the formation of a coiled coil structure, which restricts the AMP from adopting new conformations. Simulations captured the spontaneous formation of coiled coils between these peptides, and specific residues in their sequences were identified that promote unfolding. This knowledge may lead to better design of coiled coil forming peptides. Another aspect of biological membranes that can be explored with MD is the interactions between model bacterial membranes and amphipathic helices, such as the MinD membrane targeting sequence (MinD-MTS). This 11-residue helix is responsible for anchoring the MinD protein to the inner membrane of Bacillus subtilis and plays a crucial role in bacterial cell division. MinD is known to exhibit sensitivity to transmembrane potentials (TMVs), whereby its localisation and binding affinity to bacterial membranes are disrupted upon removal of the TMV. Simulations revealed rapid insertions of MinD-MTS peptides into the headgroup region of a model bacterial membrane. Analytical software was constructed to measure the membrane properties of the lipids surrounding inserted MinDMTS peptides, which revealed splayed lipid tails and suggests the MinD-MTS may be capable of inducing membrane curvature. Additional simulations were conducted to investigate the influence of a TMV on model bacterial membranes, where software was constructed to measure changes in membrane properties. An analysis of these simulations suggests that a TMV is capable of lowering the transition temperature of a model bacterial membrane by a few degrees, yielding increased fluidity in the lipids and increased perturbations on the membrane surface. Finally, another aspect of biological membranes that can be explored through MD is that of electroporation. This induction of transient water pores in cell membrane provides an exciting aspect for drug delivery applications into cells, whereby electric fields are applied to cells to increase the uptake of therapeutic drugs. Simulations of membranes with high voltage TMVs were conducted that sought to investigate the implications of electroporation across a variety of bilayer compositions at different temperatures. Software was constructed to measure changes in membrane and system properties, which revealed that pore formation occurred at the same threshold voltage for different bilayer compositions in the fluid phase (~1.9 V) and a higher voltage for DPPC bilayers in the gel phase (~2.4 V). The TMV was found to be highly dependent on the area per lipid (APL), implying that bilayers with bulkier lipids or those transitioning from gel to fluid will experience smaller TMVs and fewer pore formations. These simulations also revealed lipid flip-flopping through pores, where charged lipids tended to translocate in the direction of the electric field to produce an asymmetrically charged bilayer. Finally, simulations utilising charged peptides with membranes yielded electroporation effects, whereby the charged peptides generate an identical TMV to those produced by an ion imbalance of equal magnitude. This suggests that charged peptides, such as AMPs, may be capable of permeabilising cell membranes through electroporation mechanisms

Structure of the Vibrio cholerae Type VI secretion tubule at sub-nanometer resolution

The bacterial type VI secretion system is a multicomponent molecular machine directed against eukaryotic host cells and competing bacteria. It consists of a contractile tubule that is attached to a membrane protein complex. Upon tubule contraction, a needle is ejected into target cells to translocate toxic effectors into the cell. Due to structural and functional homologies of several proteins of the secretion system to proteins of contractile bacteriophage tails, the system is generally described as an inverted phage tail. Following this analogy, the secretion process is driven by energy stored in the elongated conformation of the Type VI secretion tubule for which also partial structural homology to bacteriophage tail sheath proteins has been predicted. However, this prediction has not been corroborated by structural data so far. The AAA+ ATPase ClpV plays an important role in the secretion process, as it disassembles the contracted tubule, putatively for recycling of the complex. Even though the binding site for ClpV has been identified in VipB, the molecular mechanism which recruits the ATPase specifically to the contracted tubule is not known yet. In a collaborative project with PD Dr. Axel Mogk and colleagues at the DKFZ Heidelberg and the group of Dr. Franz Herzog at the Gene Center Munich, we investigate the structure of the Vibrio cholerae Type VI secretion tubule consisting of the proteins VipA and VipB. We employ a hybrid methods approach of cryo electron microscopic 3D reconstruction and electron microscopic and biochemical labeling techniques supported by cross-linking mass spectrometry to develop a structural model of VipA and VipB in the tubule. We are able to resolve the three-dimensional structure of the helical VipA/B tubule up to 6 Å which allows us to locate secondary structure elements. We describe the arrangement of VipA and VipB in the asymmetric unit and show that the architecture of the tubule is mainly defined by contacts between C-terminal domains of VipB which are structurally similar to domain IV of viral tail sheath proteins. By comparison to the T4 bacteriophage tail sheath, we suggest that these structurally homologous parts mediate the common function of contraction. Additionally, the VipA/B tubule has been adapted towards efficient recycling of contracted Type VI secretion systems. VipB is equipped with a specific four-helix bundle N-terminal domain which carries the ClpV binding motif. Also for VipA, no correspondency to any other known structural part of a phage-like contractile system is found. We propose that it serves as a chaperone for VipB. Based on the observed structural homologies between the T4 phage tail sheath protein and VipB, we model the elongated state of the VipA/B tubule using known low resolution structures of the elongated T4 phage tail. Furthermore, we suggest a molecular mechanism for Type VI secretion tubule recycling. In the elongated state of the tubule, the VipB N-terminal domain is hidden in the tubule wall, making the ClpV binding motif inaccessible for the ATPase. Therefore, ClpV-mediated recycling of the tubule is restricted to its contracted state.Das bakterielle Typ-VI-Sekretionssystem ist eine aus vielen unterschiedlichen Teilen bestehende molekulare Maschine, die gegen eukaryotische Wirtszellen und konkurrierende Bakterien gerichtet ist. Sie besteht aus einem kontraktionsfähigen Tubulus, welcher mit einem Komplex aus Membranproteinen verbunden ist. Durch Kontraktion des Tubulus wird eine Nadel in eine Zielzelle gestoßen, um Gifte in die Zelle zu injizieren. Aufgrund von strukturellen und funktionalen Homologien von einigen Proteinen des Sekretionssystems zu Proteinen des kontraktionsfähigen Bakteriophagenschwanzes wird das System allgemein als umgedrehter Phagenschwanz beschrieben. In dieser Analogie wird der Sekretionsprozess durch die in der elongierten Konformation des Typ-VI-Sekretionstubulus gespeicherte Energie angetrieben. Für ihn wurde auch eine teilweise strukturelle Homologie zum Mantelprotein des Bakteriophagenschwanzes vorhergesagt, aber nie durch strukturelle Daten belegt. Die AAA+ ATPase ClpV spielt eine wichtige Rolle im Sekretionsprozess, da sie den kontrahierten Tubulus zerlegt, vermutlich zur Wiederverwertung des Komplexes. Obwohl die ClpV-Bindestelle in VipB bereits identifiziert wurde, ist der molekulare Mechanismus, der die ATPase ausschließlich an kontrahierten Tubuli binden lässt, unbekannt. In einem Kollaborationsprojekt mit PD Dr. Axel Mogk und Mitarbeitern am DKFZ Heidelberg und der Gruppe von Dr. Franz Herzog am Gen-Zentrum München, untersuchen wir die Struktur des Typ-VI-Sekretionstubulus aus Vibrio cholerae, welcher aus den Proteinen VipA und VipB besteht. Wir verbinden in unserem Ansatz die 3D-Rekonstruktion aus kryo-elektronenmikroskopischen Bildern mit elektronenmikroskopischen und biochemischen Markierungsmethoden und entwickeln ein Strukturmodell von VipA und VipB im Tubulus, welches durch den massenspektrometrischen Nachweis chemisch quervernetzter Peptide gestützt wird. Wir können die dreidimensionale Struktur des helikalen VipA/B-Tubulus bis auf 6 Å auflösen, was es uns ermöglicht, Sekundärstrukturelemente zu lokalisieren. Wir beschreiben die Anordnung von VipA und VipB in der asymmetrischen Untereinheit und zeigen, dass die Architektur des Tubulus hauptsächlich durch Kontakte zwischen C-terminalen Domänen von VipB bestimmt wird, welche strukturell der Domäne IV der Mantelproteine des Bakteriophagenschwanzes ähneln. Der Vergleich mit dem Mantel des T4 Bakteriophagenschwanzes, führt uns zu dem Vorschlag, dass diese struktur-homologen Bestandteile die gleiche Funktion in der Kontraktion besitzen. Zusätzlich ist der VipA/B-Tubulus einer effizienten Wiederverwertung des Typ-VI-Sekretionssystems angepasst. VipB besitzt eine spezielle N-terminale Domäne, die aus einem Bündel aus vier Helices besteht und das Erkennungsmotiv für ClpV trägt. Für VipA finden wir ebenfalls keine Entsprechung zu anderen phagen-ähnlichen kontraktionsfähigen Systemen. Unserer Ansicht nach dient es als Chaperon für VipB. Basierend auf den beobachteten Strukturhomologien zwischen dem Mantelprotein des T4 Bakteriophagenschwanzes und VipB, entwerfen wir unter der Verwendung von niedrig aufgelösten Strukturen des elongierten T4 Phagenschwanzes ein Modell des elongierten Zustands des VipA/B-Tubulus. Des Weiteren schlagen wir einen molekularen Mechanismus für die Wiederverwertung des Typ-VI-Sekretionstubulus vor. Im elongierten Zustand des Tubulus ist die N-terminale Domäne von VipB in der Wand des Tubulus versteckt. Daher ist das ClpV-Erkennungsmotiv für die ATPase nicht zugänglich und der Abbau des Tubulus durch ClpV auf seinen kontrahierten Zustand beschränkt

Βιοπληροφορικές μελέτες δομής και λειτουργίας μεμβρανικών πρωτεϊνών

Οι μεμβρανικές πρωτεΐνες επιτελούν µια σειρά από πολύ σημαντικές λειτουργίες, απαραίτητες για τη ζωή του κυττάρου. Τη μεγάλη πλειοψηφία των διαμεμβρανικών πρωτεϊνών, αποτελούν πρωτεΐνες των οποίων τα διαμεμβρανικά τμήματα έχουν τη δομή α-έλικας η οποία αποτελείται κυρίως από υδρόφοβα αμινοξικά κατάλοιπα που διαπερνούν το υδρόφοβο εσωτερικό της λιπιδικής διπλοστιβάδας. Η μεγάλη σπουδαιότητα των διαμεμβρανικών πρωτεϊνών, αλλά και οι εγγενείς δυσκολίες που παρουσιάζονται σε προσπάθειες κρυστάλλωσης τους, καθιστούν απαραίτητη τη δημιουργία υπολογιστικών αλγορίθμων οι οποίοι θα πρέπει να προβλέπουν σχετικά αξιόπιστα και γρήγορα τη δευτεροταγή τους δομή αλλά και τα πιθανά λειτουργικά τους χαρακτηριστικά. Αποφασιστικής σημασίας για τη μελέτη της δομής μιας διαμεμβρανικής πρωτεΐνης είναι η εύρεση της τοπολογίας της στη μεμβράνη, δηλαδή ο αριθμός των διαμεμβρανικών τμημάτων, η θέση τους στην ακολουθία της πρωτεΐνης και ο προσανατολισμός τους σε σχέση με το επίπεδο της μεμβράνης. Στα πλαίσια της διατριβής αυτής αναπτύχθηκαν υπολογιστικές μέθοδοι, βασισμένες σε σύγχρονες μαθηματικές τεχνικές, με τις οποίες θα μπορεί να γίνεται πρόγνωση της δομής και της λειτουργίας μεμβρανικών πρωτεϊνών. Συγκεκριμένα, επικεντρωθήκαμε στις α- ελικοειδείς διαμεμβρανικές πρωτεΐνες και στην κατηγορία των περιφερειακών μεμβρανικών πρωτεϊνών. Παράλληλα δημιουργήθηκαν δημόσια διαθέσιμες βάσεις δεδομένων με στοιχεία σχετικά με τη δομή και τη λειτουργία των μεμβρανικών πρωτεϊνών και ιδιαίτερων χαρακτηριστικών τους. Η μέθοδος LPLRpred που αναπτύχθηκε, επιτρέπει την εύρεση μιας περιοχής στην ακολουθία των πρωτεϊνών η οποία μπορεί να αποτελέσει θέση εισαγωγής αλληλουχιών στόχων στα πειράματα προσδιορισμού της τοπολογίας των διαμεμβρανικών πρωτεϊνών. Η πληροφορία αυτή μπορεί να χρησιμοποιηθεί για την καθοδήγηση των πειραμάτων με υπολογιστικό τρόπο και να οδηγήσει σε ελαχιστοποίηση του αριθμού και του κόστους τους. Η βάση δεδομένων ExTopoDB που κατασκευάστηκε αποτελεί την πλέον ενημερωμένη, παγκόσμια πηγή πειραματικών δεδομένων για την τοπολογία α-ελικοειδών διαμεμβρανικών πρωτεϊνών. Στις διαμεμβρανικές πρωτεΐνες οι περιοχές οι οποίες φωσφορυλιώνονται και γλυκοζυλιώνονται εδράζονται στον κυτταροπλασματικό και εξωκυττάριο χώρο αντίστοιχα. Η μέθοδος HMMpTM εισήγαγε ένα σημαντικό χαρακτηριστικό στην πρόγνωση της τοπολογίας των α-ελικοειδών διαμεμβρανικών πρωτεϊνών αξιοποιώντας αυτή την πληροφορία. Η μέθοδος GPCRpipe επιτρέπει το διαχωρισμό των συζευγμένων με G-πρωτεΐνες υποδοχέων (GPCRs) από άλλες κατηγορίες πρωτεϊνών και παρέχει σημαντικές πληροφορίες για τη δομή και λειτουργία τους. Η ανάλυση που πραγματοποιήθηκε για τις περιφερειακές μεμβρανικές πρωτεΐνες παρέχει σημαντικές πληροφορίες για τη δομή και τη λειτουργία τους στη μεμβράνη. Από τη μελέτη του δικτύου αλληλεπιδράσεων των περιφερειακών μεμβρανικών πρωτεϊνών αναδεικνύονται πρωτεΐνες οι οποίες έχουν κεντρικό ρόλο και μπορούν να αποτελέσουν στόχους της φαρμακευτικής έρευνας και περαιτέρω πειραματικής μελέτης. Η βάση mpMoRFsDB που κατασκευάστηκε, μαζί με την ανάλυση που πραγματοποιήθηκε αποτελεί την πρώτη μελέτη του φαινομένου της εγγενούς έλλειψης δομής στις αλληλεπιδράσεις των μεμβρανικών πρωτεϊνών και παρέχει σημαντικά δεδομένα για την περαιτέρω μελέτη του φαινομένου στις πρωτεΐνες αυτές.Membrane proteins perform a variety of very important biological functions necessary for the survival of the cell. The vast majority of transmembrane proteins are proteins whose transmembrane segments form an alpha-helix composed of mainly hydrophobic residues, spanning the lipid bilayer hydrophobic interior. The importance of transmembrane proteins, as well as the inherent difficulties in crystallizing and obtaining a three-dimensional structure of these proteins, dictates the need for developing computational algorithms and tools that may allow a reliable and fast prediction of their structural and functional features. In order to understand their function we must acquire knowledge about their structure and topology in relation to the membrane. By topology, we refer to the knowledge of the number and the exact localization of transmembrane segments, as well as their orientation with respect to the lipid bilayer. In this study, we developed novel algorithms and computer software, based on modern mathematical methods and machine learning approaches, to predict the structure and function of membrane proteins. We focused on two major groups; alpha-helical transmembrane proteins and peripheral membrane proteins. In addition, we constructed specialized, publicly available databases containing information about the structure and function of membrane proteins. The LPLRpred method allows the determination of a region across a protein sequence that can be used for the insertion of target sites when studying the topology of alpha-helical transmembrane proteins. This information may be used to minimize the number and the cost of experiments and computationally guide the design of new experiments. The ExTopoDB database is the most up-to-date worldwide resource including experimental information about the topology of alpha-helical transmembrane proteins. The database might be a valuable tool for researchers, in order to design new experiments and, also, for bioinformaticians, since it provides a large representative set that can be used for training and testing prediction algorithms. Phosphorylation and glycosylation are post-translational modifications (PTMs) that occur in a compartment-specific manner and therefore the presence of a phosphorylation or glycosylation site in a transmembrane protein provides valuable topological information. We examined the combination of phosphorylation and glycosylation site prediction with transmembrane protein topology prediction. The HMMpTM method integrates a novel feature in topology prediction. It is not just a consensus of post-translational modification and topology prediction but integrates in a single Hidden Markov Model phosphorylation and glycosylation prediction in order to more accurately predict the orientation of transmembrane proteins in membranes. Given that the general topology prediction algorithms perform poorly in the case of GPCRs, we developed a specialized method for their structural topological annotation, and functional classification. GPCRpipe is a pipeline for the accurate detection and annotation of GPCRs in proteomes. Moreover, GPCRpipe may offer information regarding the family of GPCRs in which the predicted proteins may belong to and the coupling specificity to certain families of G-proteins. A study of the molecular interactions of peripheral membrane proteins was performed in order to obtain insights about their role and organization, in relation to the human plasma membrane. The mpMoRFsDB database and the analysis of MoRFs in membrane proteins is the first study of such protein regions in membrane proteins and provide insights about the disorder-based protein-protein interactions in membrane proteins