A persistência bacteriana correlacionada à formação de biofilmes bacterianos é, há algum tempo, fonte de grande preocupação médica em virtude de sua ampla associação com a dificuldade de tratamento de infecções crônicas. Por outro lado, as perspectivas de utilização de biofilmes bacterianos em novas aplicações biotecnológicas e até mesmo para fins terapêuticos são promissoras. Há, portanto, grande interesse em compreender os mecanismos que levam as células bacterianas a deixar o estado planctônico, de vida livre, e associarem-se nesses conglomerados celulares altamente complexos. Ao longo das últimas décadas, o segundo mensageiro c-di-GMP – em conjunto com as moléculas que catalisam sua síntese (diguanilato ciclases) e sua degradação (fosfodiesterases) e seus receptores – estabeleceu-se como um elemento central de regulação de uma série de respostas celulares que determinam a formação ou a dispersão de biofilmes. Curiosamente, as proteínas que participam do metabolismo deste segundo mensageiro estão, frequentemente, codificadas múltiplas vezes em um mesmo genoma bacteriano. Em vista dessa observação, estudos mais recentes apontam que, para reger paralelamente uma variedade tão ampla de fenótipos, este sistema opera em modo de alta especificidade de sinalização e que, portanto, o sinal metabolizado por determinados conjuntos de diguanilato ciclases e fosfodiesterases tem alvos celulares específicos. Evidências robustas, porém isoladas até o momento, apontaram que um dos meios pelo qual ocorre a segregação entre sinal produzido e alvo específico é a interação direta entre as proteínas componentes das vias de sinalização. Mais, demonstrou-se que, em algumas vias, a transmissão de sinal ocorre exclusivamente via interação proteica, dispensando a intermediação do sinalizador em si. Para avaliar a validade e relevância global deste mecanismo, propôs-se, neste estudo, a investigação da rede total de interações entre as proteínas tipicamente associadas às vias de sinalização por c-di-GMP em Pseudomonas aeruginosa, utilizando ensaios de duplo-hibrido bacteriano. Para tanto, foram construídas duas bibliotecas de DNA direcionadas e foram feitos testes de interação de forma estratégica para possibilitar o esgotamento e averiguação de todas as possíveis interações entre as proteínas alvo identificadas. O resultado obtido, um mapa inicial, porém abrangente, da rede de interações proteicas em P. aeruginosa, indica uma grande probabilidade de que os mecanismos previamente descritos sejam realmente recorrentes e relevantes para o intermédio da sinalização nesse organismo. Algumas das interações mais robustas encontradas são bastante interessantes e serão, em estudos futuros, mais extensivamente estudadas. / Persister bacteria are correlated to biofilm formation and have been a source of great medical concern due to its close association with the impairment of traditional treatment in combating chronic infections. On the other hand, using bacterial biofilms to create original biotechnological applications or even as a means of therapeutic treatment in medical settings constitutes a promising prospect. There is, therefore, a great interest in understanding the mechanisms that allow bacteria to leave the free-living planktonic lifestyle and associate in these highly complex cellular aggregates. Over the last decades, the second messenger c-di-GMP – and also the molecules involved in its synthesis (diguanylate ciclases) and degradation (phosphodiesterases) along with its receptors – has been established as a key element implicated in regulation of a series of cellular responses that determine biofilm formation or dispersion. Curiously, the proteins that play a part in the metabolism of this second messenger are frequently coded multiple times in single bacterial genomes. Taking this into account, recent studies indicate that, in order to control such a wide range of phenotypes, this system operates via high specificity of signaling – which means that the signal metabolized by a certain set of diguanylate ciclases and phosphodiesterases has specific cellular targets. Robust but yet isolated evidence indicate that a means by which a signal is segregated with its correlated phenotypic response is through direct protein-protein interaction involving the components of these signaling pathways. Even more, there has been strikingly evidence that, in some of these pathways, signal transduction occurs exclusively through protein-protein interaction, entirely dismissing any mediation by the signal molecule. In order to validate and evaluate the global relevance of this type of mechanism, this study proposed the investigation of the entire network of interactions between proteins typically associated with c-di-GMP signaling pathways of Pseudomonas aeruginosa by employing bacterial two-hybrid system assays. To make that possible, two DNA libraries were constructed and interaction essays were performed in a strategic way so that all possibilities of interaction between target proteins were explored. The results obtained from these experiments allowed the construction of a broad map of interactions that, although still primitive, indicates that, chances are, the mechanisms previously described are both recurrent and relevant to signaling regulation in this organism. Some of the interaction partners found are particularly interesting and will be further investigated in future studies.

A proteína Cdc42 (Cell Division Cycle 42) é um membro da família das Rho GTPases, sinalizadores intracelulares conhecidos pelo seu papel na regulação do citoesqueleto. Essa proteína e capaz de ciclar entre um estado ativo (ligado à GTP) e um estado inativo (ligado à GDP) e essa ativação é modulada por diversas proteínas, conhecidas como GEFs (guanine nucleotide-exchange factors), GAPs (GTPase-activating proteins) e GDIs (guanine nucleotide-dissociation inhibitors). Trabalhos recentes têm demonstrado um papel de Cdc42 na apoptose e na senescência, respostas relacionadas e comumente desencadeadas por estresse genotóxico. Neste contexto este trabalho procurou identificar interações de Cdc42 com outras proteínas, que podem ou não estar envolvidas nos mecanismos de resposta ao dano do DNA. Para isso foram utilizadas as linhagens celulares HeLa e MRC-5 submetidas a tratamento com radiação ultravioleta tipo C, a fim de provocar danos no DNA. Foram realizados dois diferentes tratamentos em cada uma das linhagens com diferentes tempos de incubação pós radiação UV, visando a busca de proteínas envolvidas em uma resposta rápida ou tardia ao dano causado. Os lisados celulares desses tratamentos foram submetidos ao pull-down com proteínas recombinantes GST, GST-Cdc42WT (Selvagem) e GST-Cdc42V12 (Mutação constitutivamente ativa). As proteínas purificadas foram digeridas e submetidas à análise por espectrometria de massa e os dados obtidos foram utilizados para a construção de redes de interação proteica. Dentre as proteínas identificadas as que despertaram maior atenção foram: Proibitina-2 (PHB2) encontrada nas amostras incubadas por 48 horas pós irradiação e Cullina-4A (CUL4A) e P53, encontradas em amostra incubada por 5 minutos pós radiação. Essas proteínas possuem papéis em apoptose e reparo de DNA e foram observadas em posições muito próximas de Cdc42 nas redes de interação, fazendo delas interessantes alvos para futuras validações de interação proteica por análises experimentais distintas / The Cdc42 protein (Cell Division Cycle 42) is a member of the Rho family of GTPases, intracellular signalling molecules well known for their role in the cytoskeleton regulation. This protein cycles between an active state (GTP-bound) and an inactive state (GDP-bound) and this regulation is modulated by proteins known as GEFs, GAPs and GDIs. Recent studies demonstrated roles for Cdc42 in apoptosis and senescence, cellular responses commonly triggered by genotoxic stress. This work sought to identify Cdc42 interactions with other proteins that possibly involved in response to DNA damage mechanisms. To reach this aims we used HeLa and MRC-5 cell lines submitted to treatments with ultraviolet C radiation to induce DNA damage. Two experimental conditions were used in each cell line with different times and doses post UV irradiation in order to search for proteins involved in either rapid or delayed response to the installed DNA damage. Cell lysates obtained from these treatments were subjected to pull-down experiments using recombinant proteins GST, GST-Cdc42-WT (Wild type) and GST-Cdc42-V12 (constitutively active mutant). Purified proteins were digested by trypsin, analyzed by mass spectrometry and th obtained data were used for the construction of protein-protein interaction (PPI) networks. Among the identified proteins those that seem more relevant to the aims of this project were: Prohibitin-2 (PHB2), found in samples incubated 48 hours post irradiation; Cullin-4A (CUL4A) and P53, found in samples incubated 5 minutes after radiation. These proteins have roles in apoptosis and DNA repair and were observed in close proximity to Cdc42 in PPI networks, making them interesting targets for future validation by different experimental approaches

Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.

Proteins are synthesised as linear polymeric chains. The subtle energetic interplay of interatomic interactions results in chain folding, through which proteins may acquire defined structures. This spatial organisation is encoded by the protein sequence itself; the so-called thermodynamic hypothesis formulated by Anfinsen in 1961. A defined structure is often considered a pre-requisite to protein function, but widespread existence of intrinsically disordered proteins (IDPs) has prompted a re- evaluation of the ways biological function may be encoded into polypeptide chains. Furthermore, proteins often exist as part of multi-component entities, where regulation of assembly is integral to their properties. The interplay between disorder, oligomerisation and function is the focus of this thesis. Some IDPs fold conditionally upon interacting with a partner protein; a process known as coupled folding and binding. What are the biophysical advantages and consequences of disorder in the context of these interactions? A common feature of IDPs is their sequence composition bias, with charged residues being often over-represented. It is therefore tempting to speculate that electrostatic interactions may play a major role in coupled folding and binding reactions. Surprisingly, the opposite was found to be true. Charge-charge interactions only contributed about an order of magnitude to the association rate constants of two contrasting model systems. The lack of pre-formed binding interfaces-a consequence of disorder-might preclude electrostatic acceleration from complementary patches. By looking at the role of the sequence, many studies have taken a protein-centric approach to understanding disorder. Yet there is paucity of data about the effect of extrinsic factors on interactions involving disordered partners. Investigating the role of co-solutes, it was discovered that the kinetic and thermodynamic profiles of coupled folding and binding reactions were sensitive to ion-types. This effect followed the Hofmeister series, and occurred at physiological concentrations of salt. The sensitivity of coupled folding and binding reactions-a consequence of the lack of stability of IDPs-might be advantageous. Given the role of ions in biology, this 'biophysical sensing' could be a mechanism of physiological relevance, allowing modulation of protein-protein interactions involving disordered partners in response to changes in their environments. In cells, signalling networks are often multi-layered, and involve competing protein-protein interactions. The interplay between the biophysical characteristics of the components, and the behaviour of the network were investigated in a model tripartite system composed of folded and disordered proteins. The BCL-2 family regulates the intrinsic pathway of apoptosis through control of mitochondrial outer-membrane permeabilisation; a result of BAK and BAX oligomerisation. Through a shared homology motif (termed BH3), the subtle balance of their interactions determines cellular fate at the molecular level. Characterisation of the model under simple biochemical conditions revealed large differences in affinities among binary interactions; the consequence of the lifetime of the complexes, not their speed of association. A membrane-like environment, re-created using detergents, allows the oligomerisation of BAK and BAX in vitro. Furthermore, investigation of the tripartite system under detergent conditions showed that regulation of the network was the result of competing hetero- and homo-oligomerisation events. Relationships to their biophysical properties were gained by probing their energy landscapes using protein folding techniques. The connection between the biophysical properties of the components of the network and their interactions provides a molecular explanation for the regulation of apoptosis. This thesis offers insights into the ways structured assemblies and environmentally responsive disorder elements may encode functions into proteins.

Au cours des dernières années, le développement de nouvelles thérapies contre les infections bactériennes a suscité un grand intérêt face à l’émergence des nombreuses souches résistantes aux antibiotiques. Le point de départ de cette recherche de nouveaux antibiotiques, pour lesquels les bactéries n’ont pas encore acquis de mécanismes de résistance, est l’identification de nouvelles cibles cellulaires. En 2000, des études génétiques ont identifié engA, un gène bactérien dont le produit est une GTPase, comme une cible pharmacologique pertinente: elle est essentielle à la survie cellulaire, conservée au sein des bactéries et absente chez les eucaryotes.Puisque EngA agit comme un facteur d’assemblage pour le ribosome bactérien, un de nos objectifs a été de développer un test de criblage pour identifier des inhibiteurs des interactions EngA-ribosome. Ces interactions sont modulées par des changements conformationnels d'EngA, qui sont eux-mêmes déclenchés par la fixation de différents nucléotides dans le domaine catalytique. Cependant, les liens entre ces différents changements restent encore méconnus. Nous avons utilisé une approche multi-technique pour étudier ces questions et obtenir des informations utiles pour l’optimisation de notre test de criblage.Des analyses de SAXS et protéolyse limitée ont démontré un changement conformationnel en solution après adition de nucléotides di- ou tri-phosphate. La comparaison des données avec des modèles cristallographiques d'EngA a confirmé la conformation de la protéine liée au GDP. Cependant, la conformation de la protéine liée au GTP ne correspond à aucune structure connue. Des essais d’interaction ont démontré que la fixation de différents nucléotides au niveau des domaines catalytiques régule l’interaction d'EngA avec le ribosome. En outre, les effets des nucléotides se produisent en utilisant des fortes concentrations, ce qui suggère que le rôle d'EngA dans la biogenèse du ribosome peut être contrôlé par la concentration intracellulaire de nucléotides. Les travaux visant la détermination de la structure d'EngA dans sa conformation liée au GTP par cristallographie nous ont permis d’obtenir la structure d’EngA dans différentes formes cristallines. Cependant, ces structures représentent la conformation liée au GDP. L’analyse de l’empilement des cristaux a montré des contacts intermoléculaires conservés qui peuvent stabiliser cette conformation pendant la nucléation. Des mutations spécifiques permettant la rupture de ces contacts peuvent éventuellement aider à promouvoir la cristallisation de conformations alternatives. Des analyses de cryo-microscopie électronique ont débuté afin d’obtenir la structure du complexe EngA:50S de chez B. subtilis. Des résultats préliminaires montrent une carte de densité électronique à 6.4 Å de résolution. L’interprétation de ces résultats est en cours. / The development of new therapeutics against bacterial infections has aroused great interest over the last years in the context of drug resistance. The starting-point in the pursuit of new antibiotics for which bacterial resistance mechanisms do not exist is the identification of novel cellular targets. Genetics studies in the early 2000s have identified engA as a conserved bacterial gene whose product is a GTPase that could represent a potential drug target: it is conserved among bacteria, essential for cell survival, and absent in humans.Since EngA acts as an assembly factor for the bacterial ribosome, one of our aims was to develop an assay to screen inhibitors of the EngA-ribosome interactions. These interactions are modulated by EngA conformational changes that are in turn triggered by the binding of different nucleotides to the catalytic G-domain. As the interplay between all these events in bacteria is still not resolved, we have used a multi-technique approach to explore these questions in order to obtain useful information for the setting up of a robust screening assay.SAXS and limited proteolysis showed a conformational change occurring in solution upon addition of either di- or tri-phosphate nucleotides. While model validation analysis confirmed the GDP-bound conformation, the GTP-bound state does not match any known EngA structure. Binding studies have revealed modulation of interactions by different nucleotide-bound states. Furthermore, response to nucleotides occurs at high concentrations, suggesting that the role of EngA in promoting ribosome assembly could be monitored by the intracellular nucleotide concentration. Efforts on identifying the GTP-bound state 3D structure by crystallography have resulted in EngA structures in different crystal forms. Although all the obtained structures represent the GDP-bound state, packing analysis has revealed conserved crystal contacts that can potentially stabilise this conformation during nucleation. Specific mutations aiming at disrupting these contacts may help to promote crystallisation of alternative conformations. Cryo-EM investigation has been initiated in order to obtain the structure of the B. subtilis EngA:50S complex. So far, an electron density map at 6.4 Å resolution has been obtained and its interpretation is underway.

With the advent of large scale genome sequencing efforts along with more sophisticated methods of genetic mapping, a number of loci have been identified that are associated with human diseases. Intriguingly, many genes identified in these loci remain uncharacterized. Although current annotation can provide a prediction of putative function of some of these proteins at a biochemical level, understanding their cellular roles require analysis at a single gene level. Bioinformatic analysis carried out in the laboratory during studies on cyclic nucleotide metabolism in mycobacteria identified putative Class III cyclic nucleotide phosphodiesterases (Class III cNMP PDEs) from the non-redundant database of proteins. One of the proteins identified was the Rv0805 gene product from Mycobacterium tuberculosis. Detailed biochemical characterization of this protein revealed that Rv0805 is indeed a phosphodiesterase (PDE) and could hydrolyze 3’, 5’-cyclic adenosine monophosphate (cAMP) as well as 3’, 5’-cyclic guanosine monophosphate (cGMP). Structural analysis of Rv0805 revealed a metallophosphoesterase (MPE) like fold and presence of two metal atoms at the binuclear metal centre of the protein. Moreover, overexpression of Rv0805 in E. coli and M. smegmatis reduced intracellular cAMP levels indicating that it possesses cAMP PDE activity in vivo. The majority of proteins identified in this bioinformatic analysis were of bacterial or archaebacterial in origin but it was interesting to find some mammalian proteins, since, till date, no Class III cNMP PDE has been found in higher eukaryotes. Interestingly, two genes were identified in the human genome. These genes, 239FB and 239AB, are expressed in the fetal brain and adult brain, respectively and have been annotated as metallophosphoesterases but there has been no biochemical or functional characterization of these proteins. The 239FB gene is present between the FSHB and PAX6 genes on chromosome 11. This gene locus is present within a deletion interval (11p13-14) that is associated with the mental retardation phenotype of WAGR syndrome (Wilms’ tumor, aniridia, genitourinary anomalies, mental retardation). Inspection of available sequenced mammalian genomes indicated a shared synteny of the genes in the WAGR locus, highlighting it’s evolutionary conservation. Most interestingly, nucleotide sequences within the WAGR locus (which include the 5 genes WT1, PAX6, RCN1, ELP4 and 239FB) are amongst the 481 ultra conserved regions of the human genome. Moreover, 239FB is one of only 24 instances where an ortholog of an ultra-conserved element could be partially traced back by sequence similarity in lower eukaryotes such as Ciona intestinalis, Drosophila melanogaster, or Caenorhabditis elegans. Although the function of the 239FB protein is unknown so far, the distinctive expression of the gene in the fetal brain and the presence of an “ancient conserved region” in this gene suggest that this gene may be vital for the development of the nervous system. The work carried out in this thesis has attempted to understand the physiological functions of the 239FB/AB gene family. Amino acid sequence comparison revealed two amino acids changes between the human and rat proteins indicating the extra-ordinary sequence conservation of these proteins. Therefore, to characterize the biochemical properties of 239FB and 239AB proteins, rat proteins were used as model enzymes. Reverse transcription-PCR analysis of RNA prepared from the fetal and adult rat brains as well as Western blot analysis on cytosolic fractions of rat brains from various developmental stages indicated that 239FB is predominantly expressed in fetal brain. Detailed biochemical analyses of the rat 239FB and 239AB proteins were performed which showed that they possess metallophosphodiesterase activity. 239FB showed activity only in the presence of Mn2+ and Co2+ as the added metal cofactors. Surprisingly, the Km for Mn2+ of 239FB was found to be 1.5 mM, which is nearly 60-fold higher than that of its mycobacterial ortholog, Rv0805. A systematic mutational analysis was performed to characterize the residues that are involved in binding either one or both the metals found in the catalytic site of 239FB. Although 239FB shares some of the residues that have been shown to be essential for metal binding and catalytic activity with other MPEs including Rv0805, there are some differences as well. One histidine residue that has been conserved in other MPEs and has been shown to be important for metal binding is replaced by glycine (Gly-252) in 239FB. To study the consequence of replacing the glycine with a histidine in 239FB, a 239FBGly252His mutant protein was generated and characterized. Interestingly, the single mutation of Gly-252 to a histidine residue not only increased the affinity of the protein for metals but increased catalytic activity as well with various phosphodiesters. Moreover, 239FBGly252His mutant protein showed significant activity with cAMP and cGMP which were not hydrolysed by wild type 239FB. Interestingly, in the 239AB protein, histidine 284 is present at a position equivalent to Gly-252 in the 239FB protein. Biochemical characterization of 239AB showed 2’, 3’-cAMP hydrolyzing activity similar to 239FBGly252His mutant protein. A rat 239FB protein with a mutation (His67Arg) corresponding to a single nucleotide polymorphism seen in human 239FB, led to complete inactivation of the protein. The occurrence of this SNP at a very low frequency and only as a heterozygous condition suggests that a complete loss-of-function mutation of 239FB in human populations cannot be tolerated. To gain insights into the function of 239FB in its physiological milieu, yeast two-hybrid screening was performed with 239FB using human fetal brain cDNA library. Dipeptidyl peptidase III, a zinc dependent metallopeptidase, was found as an interacting partner of 239FB in this analysis and the functional consequences of this interaction would be an interesting area of study in future. While a number of metallophosphoesterases have been characterized biochemically and structurally, their biological role(s) and in vivo substrate(s) remain elusive. In order to elucidate the physiological role of 239FB/AB family, the ortholog of 239FB/AB in D. melanogaster was characterized. Sequence comparison of Drosophila ortholog with both the mammalian proteins indicated that it may be an ortholog of 239AB and hence, it was named as d239AB. Enhancer-promoter analysis with a putative promoter region of the d239AB indicated the expression of d239AB in the mushroom bodies in brain and in enterocytes in mid gut. Characterization of a Drosophila line, BS#16242, with a piggybac element inserted in the intron of d239AB showed disruption of d239AB expression. This suggested that BS#16242 line can serve as a d239AB knockout line and hence, was selected for further phenotypic characterization to unravel the physiological roles of d239AB. Though, BS#16242 flies did not show any developmental defects, a severe reduction in the fecundity of these files was observed. Further analysis revealed defective ovulation as a probable reason for reduced fecundity of these flies. In addition to compromised fecundity, BS#16242 flies showed a significant reduction in the life span of male as well as female flies. Moreover, these flies showed less resistance to thermal stress and desiccation. Most interestingly, all these phenotypes were rescued upon neuronal expression of the d239AB transgene in BS#16242 flies indicating that neuronal function of d239AB is important for diverse physiological processes. The phenotypes observed in BS#16242 flies mimic the physiological state under increased insulin signaling, such as decrease in life span, and susceptibility to various stress conditions suggesting that d239AB could play a role in the insulin signaling pathway. Interestingly, overexpression of d239AB transgene in neurons reduced cAMP levels in the brains of Drosophila, indicating that the protein may have cAMP phosphodiesterase activity in vivo. This is the first analysis of the presence of a Class III phosphodiesterase in eukaryotes. Thus, d239AB mediated regulation of cAMP levels in a particular subsets of cells, such as neurons, could also be one of the molecular mechanisms responsible for reduced fecundity and longevity of BS#16242 flies. Interacting partners of d239AB were inspected in the Drosophila interactome (built on protein-protein interactions identified using a yeast two-hybrid approach). Strikingly, most of the d239AB interacting proteins were involved either in transcriptional or translational regulation indicating that d239AB could be involved in the regulation of expression of genes involved in diverse physiological processes. This could explain why disruption of d239AB led to various physiological defects such as reduced fecundity, decreased life span and compromised fitness. In summary, studies described in this thesis suggest that 239FB and 239AB proteins are the first Class III cyclic nucleotide phosphodiesterases reported in eukaryotes. Results shown here suggest the critical role of their ortholog in the physiology of Drosophila. Further genetic manipulation in D. melanogaster and other organisms which harbor orthologs of the 239FB/AB gene could throw light on the diverse biological roles of these enzymes in humans.

Protein-protein interactions (PPIs) are attractive targets because of their therapeutic potential. One approach to design small molecules that can disrupt the PPIs is to use structural information of proteins. With this approach, triazole-based peptidomimetics that mimic beta-turn hot-spot regions in neurotrophins were synthesized. The monovalent mimics were assembled into bivalent mimics via a combinatorial method. Three different bivalent mimics were prepared for different studies. Bivalent mimics with long-linkers bound to TrkA or TrkC receptor and showed partial antagonism for the receptors. Other mimics were conjugated with cytotoxic compounds and they were used for TrkC targeted drug delivery. The last group of bivalent mimics previously showed targeted delivery effects for pancreatic cancer cells. In this study, we synthesized Eu-chelated bivalent mimics to perform a competitive binding assay for pancreatic cancer cells. Previous research in our group focused on design of secondary structures' mimics on rigid scaffolds as "minimalist mimics." We sought to establish structural design criteria for the minimalist mimics, and we wanted to propose that sets of such compounds could mimic local pairs of amino acids in any secondary structures as "universal peptidomimetics." Thus, we designed five compounds, such as oxazoline-, pyrrole-, dyine- "kinked" and "linear" bistrizole-based peptidomimetics, and performed molecular modelings, DFT calculations, and QMD for them to validate our hypothesis. On the concepts of "minimalist mimics" and "universal peptidomimetics," we developed the C alpha ? C beta vector matching program to evaluate preferred orientations of C alpha - C beta coordinates for secondary structures. We applied the program to omegatides and pyrrolinone-pyrrolidine oligomers. The compounds matched better with strands than for helices. We expanded the C alpha ? C beta vector matching idea to a method that ranks preferred conformations of small molecules on any combination of three interface side-chains in all structurally characterized PPIs. We developed a PDB mining program (explores key orientation, EKO) to do this, and EKO applied to pyrrolinone-pyrrolidine oligomers to find targets. EKO found several interesting targets, such as AICAR Tfase, GAPDH, and HIV-1 protease. HIV-1 dimerization inhibition and Zhang-Poorman kinetic assays were performed to validate our hypothesis, and the results showed that pyrrolinone-pyrrolidine derivatives inhibited HIV-1 dimerization.

BACKGROUND: A long-standing challenge in the post-genomic era of Bioinformatics is the prediction of protein-protein interactions, and ultimately the prediction of protein functions. The problem is intrinsically harder, when only amino acid sequences are available, but a solution is more universally applicable. So far, the problem of uncovering protein-protein interactions has been addressed in a variety of ways, both experimentally and computationally. MOTIVATION: The central problem is: How can protein complexes with solved threedimensional structure be utilized to identify and classify protein binding sites and how can knowledge be inferred from this classification such that protein interactions can be predicted for proteins without solved structure? The underlying hypothesis is that protein binding sites are often restricted to a small number of residues, which additionally often are well-conserved in order to maintain an interaction. Therefore, the signal-to-noise ratio in binding sites is expected to be higher than in other parts of the surface. This enables binding site detection in unknown proteins, when homology based annotation transfer fails. APPROACH: The problem is addressed by first investigating how geometrical aspects of domain-domain associations can lead to a rigorous structural classification of the multitude of protein interface types. The interface types are explored with respect to two aspects: First, how do interface types with one-sided homology reveal convergently evolved motifs? Second, how can sequential descriptors for local structural features be derived from the interface type classification? Then, the use of sequential representations for binding sites in order to predict protein interactions is investigated. The underlying algorithms are based on machine learning techniques, in particular Hidden Markov Models. RESULTS: This work includes a novel approach to a comprehensive geometrical classification of domain interfaces. Alternative structural domain associations are found for 40% of all family-family interactions. Evaluation of the classification algorithm on a hand-curated set of interfaces yielded a precision of 83% and a recall of 95%. For the first time, a systematic screen of convergently evolved motifs in 102.000 protein-protein interactions with structural information is derived. With respect to this dataset, all cases related to viral mimicry of human interface bindings are identified. Finally, a library of 740 motif descriptors for binding site recognition - encoded as Hidden Markov Models - is generated and cross-validated. Tests for the significance of motifs are provided. The usefulness of descriptors for protein-ligand binding sites is demonstrated for the case of "ATP-binding", where a precision of 89% is achieved, thus outperforming comparable motifs from PROSITE. In particular, a novel descriptor for a P-loop variant has been used to identify ATP-binding sites in 60 protein sequences that have not been annotated before by existing motif databases.

<p>Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the Z_SPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: Z_Taq, originally selected to bind Taq DNA polymerase, and anti-Z_Taq, an anti-idiotypic binder to Z_Taq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free Z_SPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Ų total surface area and 10 out of 13 varied residues in Z_SPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of Z_SPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free Z_SPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of Z_SPA-1 upon binding seriously reduces the binding affinity. The Z_Taq:anti-Z_Taq complex buries in total 1672 Ų surface area and all varied positions in anti-Z_Taq are directly involved in binding. The main differences between the Z:Z_SPA-1 and the Z_Taq:anti-Z_Taq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither Z_Taq nor anti-Z_Taq shows the molten globule behaviour seen for Z_SPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.</p>

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.