Non-catalytic protein-carbohydrate interactions are an essential element of various biological events. This dissertation presents the work on understanding carbohydrate recognition mechanisms and their physical significance in two groups of non-catalytic proteins, also called lectins, which play key roles in major applications such as cellulosic biofuel production and drug delivery pathways. A computational approach using molecular modeling, molecular dynamic simulations and free energy calculations was used to study molecular-level protein-carbohydrate and protein-protein interactions. Various microorganisms like bacteria and fungi secret multi-modular enzymes to deconstruct cellulosic biomass into fermentable sugars. The carbohydrate binding modules (CBM) are non-catalytic domains of such enzymes that assist the catalytic domains to recognize the target substrate and keep it in proximity. Understanding the protein-carbohydrate recognition mechanisms by which CBMs selectively bind substrate is critical to development of enhanced biomass conversion technology. We focus on CBMs that target both oligomeric and non-crystalline cellulose while exhibiting various similarities and differences in binding specificity and structural properties; such CBMs are classified as Type B CBMs. We show that all six cellulose-specific Type B CBMs studied in this dissertation can recognize the cello-oligomeric ligands in bi-directional fashion, meaning there was no preference towards reducing or non-reducing end of ligand for the cleft/groove like binding sites. Out of the two sandwich and twisted forms of binding site architectures, twisted platform turned out to facilitate tighter binding also exhibiting longer binding sites. The exterior loops of such binding sites were specifically identified by modeling the CBMs with non-crystalline cellulose showing that high- and low-affinity binding site may arise based on orientation of CBM while interacting with non-crystalline substrate. These findings provide various insights that can be used for further understanding of tandem CBMs and for various CBM based biotechnological applications. The later part of this dissertation reports the identification of a physiological ligand for a mammalian glycoprotein YKL-40 that has been only known as a biomarker in various inflammatory diseases and cancers. It has been shown to bind to oligomers of chitin, but there is no known function of YKL-40, as chitin production in the human body has never been reported. Possible alternative ligands include proteoglycans, polysaccharides, and fibers such as collagen, all of which make up the mesh comprising the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. We considered the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. Our simulation results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, as the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non- specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to the associated increases in hyaluronan.

Kohlenhydrate stellen aufgrund der strukturellen Vielfalt und ihrer oft exponierten Lage auf Zelloberflächen wichtige Erkennungsstrukturen dar. Die Wechselwirkungen von Proteinen mit diesen Kohlenhydraten vermitteln einen spezifischen Informationsaustausch. Protein-Kohlenhydrat-Interaktionen und ihre Triebkräfte sind bislang nur teilweise verstanden, da nur wenig strukturelle Daten von Proteinen im Komplex mit vorwiegend kleinen Kohlenhydraten erhältlich sind. Mit der vorliegenden Promotionsarbeit soll ein Beitrag zum Verständnis von Protein-Kohlenhydrat-Wechselwirkungen durch Analysen struktureller Thermodynamik geleistet werden, um zukünftig Vorhersagen mit zuverlässigen Algorithmen zu erlauben. Als Modellsystem zur Erkennung komplexer Kohlenhydrate diente dabei das Tailspike Protein (TSP) aus dem Bakteriophagen HK620. Dieser Phage erkennt spezifisch seinen E. coli-Wirt anhand der Oberflächenzucker, der sogenannten O-Antigene. Dabei binden die TSP des Phagen das O-Antigen des Lipopolysaccharids (LPS) und weisen zudem eine hydrolytische Aktivität gegenüber dem Polysaccharid (PS) auf. Anhand von isolierten Oligosacchariden des Antigens (Typ O18A1) wurde die Bindung an HK620TSP und verschiedener Varianten davon systematisch analysiert. Die Bindung der komplexen Kohlenhydrate durch HK620TSP zeichnet sich durch große Interaktionsflächen aus. Durch einzelne Aminosäureaustausche im aktiven Zentrum wurden Varianten generiert, die eine tausendfach erhöhte Affinität (KD ~ 100 nM) im Vergleich zum Wildtyp-Protein (KD ~ 130 μM) aufweisen. Dabei zeichnet sich das System dadurch aus, dass die Bindung bei Raumtemperatur nicht nur enthalpisch, sondern auch entropisch getrieben wird. Ursache für den günstigen Entropiebeitrag ist die große Anzahl an Wassermolekülen, die bei der Bindung des Hexasaccharids verdrängt werden. Röntgenstrukturanalysen zeigten für alle TSP-Komplexe außer für Variante D339N unabhängig von der Hexasaccharid-Affinität analoge Protein- und Kohlenhydrat-Konformationen. Dabei kann die Bindestelle in zwei Regionen unterteilt werden: Zum einen befindet sich am reduzierenden Ende eine hydrophobe Tasche mit geringen Beiträgen zur Affinitätsgenerierung. Der Zugang zu dieser Tasche kann ohne große Affinitätseinbuße durch einen einzelnen Aminosäureaustausch (D339N) blockiert werden. In der zweiten Region kann durch den Austausch eines Glutamats durch ein Glutamin (E372Q) eine Bindestelle für ein zusätzliches Wassermolekül generiert werden. Die Rotation einiger Aminosäuren bei Kohlenhydratbindung führt zur Desolvatisierung und zur Ausbildung von zusätzlichen Wasserstoffbrücken, wodurch ein starker Affinitätsgewinn erzielt wird. HK620TSP ist nicht nur spezifisch für das O18A1-Antigen, sondern erkennt zudem das um eine Glucose verkürzte Oligosaccharid des Typs O18A und hydrolysiert polymere Strukturen davon. Studien zur Bindung von O18A-Pentasaccharid zeigten, dass sich die Triebkräfte der Bindung im Vergleich zu dem zuvor beschriebenen O18A1-Hexasaccharid verschoben haben. Durch Fehlen der Seitenkettenglucose ist die Bindung im Vergleich zu dem O18A1-Hexasaccharid weniger stark entropisch getrieben (Δ(-TΔS) ~ 10 kJ/mol), während der Enthalpiebeitrag zu der Bindung günstiger ist (ΔΔH ~ -10 kJ/mol). Insgesamt gleichen sich diese Effekte aus, wodurch sehr ähnliche Affinitäten der TSP-Varianten zu O18A1-Hexasaccharid und O18A-Pentasaccharid gemessen wurden. Durch die Bindung der Glucose werden aus einer hydrophoben Tasche vier Wassermoleküle verdrängt, was entropisch stark begünstigt ist. Unter enthalpischen Aspekten ist dies ebenso wie einige Kontakte zwischen der Glucose und einigen Resten in der Tasche eher ungünstig. Die Bindung der Glucose in die hydrophobe Tasche an HK620TSP trägt somit nicht zur Affinitätsgenerierung bei und es bleibt zu vermuten, dass sich das O18A1-Antigen-bindende HK620TSP aus einem O18A-Antigen-bindenden TSP evolutionär herleitet. In dem dritten Teilprojekt der Dissertation wurde der Infektionsmechanismus des Phagen HK620 untersucht. Es konnte gezeigt werden, dass analog zu dem verwandten Phagen P22 die Ejektion der DNA aus HK620 allein durch das Lipopolysaccharid (LPS) des Wirts in vitro induziert werden kann. Die Morphologie und Kettenlänge des LPS sowie die Aktivität von HK620TSP gegenüber dem LPS erwiesen sich dabei als essentiell. So konnte die DNA-Ejektion in vitro auch durch LPS aus Bakterien der Serogruppe O18A induziert werden, welches ebenfalls von dem TSP des Phagen gebunden und hydrolysiert wird. Diese Ergebnisse betonen die Rolle von TSP für die Erkennung der LPS-Rezeptoren als wichtigen Schritt für die Infektion durch die Podoviren HK620 und P22. / Carbohydrates are important for recognition events because of their diverse structure and their exposition on cell surfaces. Interactions between proteins and carbohydrates mediate a specific exchange of information crucial for manifold biological functions. The energetics of protein-carbohydrate-interactions are not very well understood so far due to the lack of structural data of proteins in complex with extensive oligosaccharides consisting of more than two building blocks. This dissertation improves the understanding of how proteins recognize complex carbohydrates by analysis of structural thermodynamics, which might lead to reliable algorithms for predictions of protein-carbohydrate-interactions. As model system for this work the tailspike protein (TSP) from coliphage HK620 was used. This phage recognizes specifically the surface O-antigen of its E. coli host by its TSP. HK620TSP does not only bind the O-antigen of host lipopolysaccharide (LPS), but also cleaves the polysaccharide (PS) by its endo-N-acetylglusaminidase activity. HK620TSP binds hexasaccharide fragments of this PS with low affinity (KD ~ 130 μM). However, single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants (KD ~ 100 nM). Strikingly, at room temperature association is driven by enthalpic and entropic contributions emphasizing major solvent rearrangements upon complex formation. Regardless of their affinity towards hexasaccharide the TSP complexes showed only minor conformational differences in crystal structure analysis accept of mutant D339N. The extended sugar binding site can be subdivided into two regions: Firstly, there is a hydrophobic pocket at the reducing end with minor affinity contributions. Surprisingly, access to this site is blocked by a single exchange of aspartate to asparagine (D339N) without major loss in hexasaccharide affinity. Secondly, there is a region where specific exchange of glutamate for glutamine (E372Q) creates a site for an additional water molecule. Upon sugar binding side chain rearrangements lead to displacement of this water molecule and additional hydrogen bonding. Thereby this region of the binding site is defined as the high affinity scaffold. HK620TSP is not only specific for the O18A1-antigen, but also the lacking of the branching glucose in the O18A1-antigen can be tolerated so that the accordant O18A PS can be bound and cleaved by HK620TSP as well. Surprisingly, in binding studies with the smallest O-antigen units of these PS the O18A pentasaccharide was bound by TSP variants with nearly the same affinity or even a slightly increased one compared to the O18A1 hexasaccharide. However, there is a change in thermodynamic contributions to binding: the lack of the glucose moiety leads to a less entropically favored binding compared to binding of O18A1-hexasaccharide (Δ (-TΔS) ~ 10 kJ/mol). In contrast the enthalpic contribution to the binding is more favorable (ΔΔH ~ -10 kJ/mol) for the binding of O18A pentasaccharide. The side-chain glucose contributes to entropy by the release of four water molecules out of a hydrophobic pocket. The binding of this branching glucose is paid by an enthalpic penalty because of the breakup of hydrogen bonding of displaced water molecules and destabilizing contacts between sugar and protein in this hydrophobic pocket. Therefore the binding of the glucose in this pocket does not account for generating affinity and an evolutionary relation of HK620TSP to an O18A-antigen binding protein is presumed. Finally, the infection mechanism of phage HK620 was studied as well. In analogy to the related phage P22 the DNA-ejection could be triggered by incubation of HK620 with the host LPS in vitro. The morphology and chain length of the LPS as well as the activity of HK620TSP towards the LPS are crucial for this in vitro DNA-ejection. Thus, the DNA-ejection could also be induced by LPS from bacteria of serogroup O18A which can be bound and hydrolyzed by HK620TSP. These results stress the role of TSP for the recognition of host LPS-receptors as a crucial step of infection by podoviruses P22 and HK620.

Protein-carbohydrate interactions are pivotal to many biological processes, from plant cell wall degradation to host-pathogen interactions. Many of these processes require the deployment of carbohydrate-active enzymes in order to achieve their intended effects. One such class of enzymes, glycoside hydrolases, break down carbohydrate substrates by hydrolyzing the glycosidic bond within polysaccharides or between carbohydrates and non-carbohydrate moieties. The catalytic efficiency of glycoside hydrolases is often enhanced by carbohydrate-binding modules (CBMs) which are part of the modular structure of these enzymes. Understanding the carbohydrate binding function of these modules is often key to studying the catalytic properties of the enzyme. This thesis investigates the molecular determinants of carbohydrate recognition by CBMs that share similar amino acid sequences and overall three-dimensional structures and thus fall within the same CBM family. Specifically this research focused on two families; plant cell wall binding family 6 CBMs and the alpha-glucan binding family 41 CBMs. Through X-ray crystallography, isothermal titration calorimetry and other biochemical experiments, the structural and biophysical properties of CBMs were analyzed. Studying members of CBM family 6 allowed us to establish the overall picture of how similar CBMs interact with a diverse range of polysaccharide ligands. This was found to be due to changes in the topology of the binding site brought about by changes in amino acid side chains in very distinct regions of the binding pocket such that it adopted a three-dimensional shape that is complementary to the shape of the carbohydrate ligand. Members of CBM family 41 were shown to have nearly identical modes of starch recognition as found in starch-binding CBMs from other families. However family 41 CBMs are distinct as they are found mainly in pullulanases (starch debranching enzymes) and have developed binding pockets which are able to accommodate alpha-1,6-linkages, unlike other starch-binding CBM families. These are the first studies comparing multiple CBMs from within a given CBM family at the molecular level whose results allow us to examine the distinct modes of carbohydrate recognition within a CBM family. Analysis of the family 41 CBMs revealed that these CBMs are mainly found in pullulanases from pathogenic bacteria. Members from Streptococcal species were shown to specifically interact with glycogen stores within mouse lung tissue, leading us to investigate the role of alpha-glucan degradation by the pullulanase SpuA in the pathogenesis of Streptococcus pneumoniae. SpuA targets the alpha-1,6-branches in glycogen granules, forming alpha-1,4-glucan products of varying lengths. The overall three-dimensional structure of SpuA in complex with maltotetraose was determined by X-ray crystallography and showed that its active site architecture is optimal for interacting with branched substrates. Additionally, the N-terminal CBM41 module participates in binding substrate within the active site, a novel feature for CBMs. This is the first study of alpha-glucan degradation by a streptococcal virulence factor and aids in explaining why it is crucial for full virulence of the organism.

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