DNA nucleases are essential for various biological functions such as replication, recombination, and repair. Restriction endonucleases (REs) are excellent model system for the investigation of DNA recognition and specificity. SgrAI is a type IIF RE that cuts an 8 base pair primary sequence. In addition to its primary cleavage activity it also cleaves secondary sequences, but only appreciably in the presence of the primary sequence. The longer flanking DNA exhibits much greater activated DNA cleavage by SgrAI (>1000 fold activation by secondary site). Interestingly, the asymmetric cleavage seen in one of the two types of secondary site DNA is lost upon activation of SgrAI, suggesting a loss of communication between DNA recognition and activity upon specificity expansion. The structure of SgrAI bound to 22-1HT supports the cryoelectron microscopy structure of activated, oligomeric SgrAI highlighting the significance of the contacts made by the flanking DNA and the role played by N-terminal domain contacts in forming the run-on oligomer. The biological study suggests that the run-on oligomer formation sequesters the host DNA from being cleaved by the activated SgrAI complex. The DNA sequence binding, cleavage preference, and the structure of K96A SgrAI were determined. Unexpectedly, this mutation did not alter the structure of the enzyme, nor did it result in an enzyme lacking sequence preference at the 7ᵗʰ position. Instead, the largest effect of the mutation appears to be in making the enzyme more specific such that it fails to cleave either type of secondary site. It may be that the K96 side chain is required to distort the non YG sequences (specifically GG and TC) of secondary site DNA for proper positioning in the enzyme active site upon activation and specificity expansion. The crystal structure of Mk0566, XPG homologue from M. kandleri, was solved to 2.48 Å resolution and was found to be very similar to that of human FEN-1 and to other archaeal FEN-1/XPG homologues. These results suggest that the main biological role of Mk0566 is in DNA replication; however, they do not preclude involvement in a modified form of nucleotide excision repair.

L'acide rétinoïque (AR) joue un rôle important dans plusieurs processus cellulaires à travers la régulation de la différentiation cellulaire, de la prolifération et de l'apoptose. Ces propriétés sont à la base de l'utilisation de l'AR dans le traitement de plusieurs cancers dont la leucémie aiguë promyélocytaire. Décrypter comment l'AR contrôle l'expression de gènes spécifiques est un défi permanent pour l'étude des cancers. Les effets de l'AR sont médiés in vivo principalement par les récepteurs à l'acide rétinoïque (RARs). Il a été récemment démontré que la phosphorylation des RARs par différentes kinases est une étape nécessaire dans la régulation de leurs fonctions. Dans ce contexte, ma thèse a porté sur l’étude des mécanismes moléculaires de la régulation par phosphorylation des RARs. Nous nous sommes intéressés en particulier à deux aspects : l’effet de la phosphorylation sur le domaine de liaison au ligand (LBD) et sur le domaine N-terminal (NTD). Dans le cas du LBD, la phosphorylation induit la fixation de la Cycline H qui est une sous-unité du facteur de transcription TFIIH, alors que la phosphorylation du NTD induit une diminution d’affinité de liaison à la Vinexine beta qui est un co-répresseur. Nous avons étudié les effets de la phosphorylation par des simulations de dynamique moléculaire. Cette technique permet de caractériser la dynamique structurale et de quantifier les interactions qui stabilisent les états phosphorylés et non phosphorylés. Ce projet a permis de définir les bases moléculaires de la communication entre le RA et les cascades de phosphorylation et d’obtenir des informations originales sur des mécanismes régulateurs d’une grande importance. / Retinoic Acid (RA) plays a critical role in many cellular processus through regulatory effects on cellular differentiation, proliferation and apoptosis. This proprety is at the basis of RA therapy in the treatment of several diseases and cancers such as Acute Promyelocytic Leukemia. Deciphering how RA controls the expression of specific subsets of genes is therefore a permanent challenge in oncology. The effects of RA are mediated in vivo by the retinoic acid receptor (RAR), which consistsof three subtypes. A new concept has recently emerged according to which phosphorylation of RARs by different kinases is a necessary step in the regulation of their function. In this context, the specific aim of this thesis was the elucidation of the molecular mechanisms of the regulation of RAR mediated by phosphorylation. In particular, we focused on two aspects, the effects of phosphorylation of the ligand binding domain (LBD) and the effects on the N-terminal domain (NTD). In the case of the LBD, phosphorylation enhanced binding to cyclin H, a component of the TFIIH transcription factor, while phosphorylation of the NTD decreased binding to vinexinB, a corepressor protein. We used molecular dynamics simulations to characterize the structural dynamics of these proteins in both phosphorylated and unphosphorylated states and to quantify theirinteractions. From this project, we were able to define the molecular basis of the communication between RA-induced phosphorylation cascades and regulatory mechanisms of high importance.

Although the regulation of proteins functions by allosteric interactions has been identified in many subcellular processes, long-range conformational changes in proteins are also known to be induced by molecular switches. A molecular switch based on the cis-trans isomerization of a peptidyl-prolyl bond is capable of inducing a conformational change directly to the protein backbone, which is then propagated throughout the system. However, these switches are elusive and difficult to identify due to their intrinsic dynamics in the biomolecules where they are found. Herein, we explore the conformational dynamics and free energy landscape of the SH2 domain of Interleukin-2-inducible T-Cell Kinase (ITK) to fully understand the conformational coupling between the distal cis-trans molecular switch, and its phosphotyrosine binding pocket. Using multiple microsecond-long all-atom molecular dynamics simulations in explicit water for over a total of 60 μs, we show that the cis-trans isomerization of the Asn286-Pro287 peptidyl-prolyl bond is directly correlated to the dynamics of the phosphotyrosine binding pocket, in agreement with previous NMR studies. While the cis state is localized to a single free energy basin and less dynamic, the trans state samples two distinct conformations of its binding pocket – one that recognizes the phosphotyrosine motif, and another that is similar the cis state. These results provide an atomic-level description of a less-well understood allosteric regulation by a peptidyl-prolyl cis-trans molecular switch that could aid in the understanding of normal and aberrant sub-cellular process and the identification of these elusive molecular switches in other proteins.

No description available.

Phenylalanine hydroxylase (PAH, EC: 1.14.16.1) is a non-heme iron tetrahydropterin-dependent monooxygenase that maintains phenylalanine (L-Phe) homeostasis via conversion of L-Phe to L-Tyr. PAH is an allosteric enzyme that converts from an inactive T-state to an active R-state upon addition of substrate, L-Phe. Allosteric activation is correlated with physical and structural changes within the enzyme and a large activation energy. Crystal structures of PAH have not identified the location of the allosteric effector binding site. Herein, we report computational protein mapping efforts using the FTmap algorithm and experimental site-directed mutagenesis studies designed to define and screen possible L-Phe allosteric binding sites. Mass spectroscopic analysis of PAH proteolytic fragments obtained after photo-crosslinking with 2-azido-3-phenylpropanoate overlapped with one computationally derived allosteric binding pocket containing residues 110-120 and 312-317. Ligand docking studies, fluorescence measurements, binding affinity and activity assays on wild-type and mutant enzymes further characterized the shape and specificity of this pocket. Thermodynamic studies using surface acoustic wave (SAW) biosensing determined the affinity of L-Phe for the allosteric site. Two L-Phe binding sites were observed upon SAW titrations, corresponding to the active and allosteric sites respectively ( K D,app^on 113 ± 12 µM active site, K D,app^on 680 ± 20 µM allosteric site). Site-directed mutagenesis was performed to prepare mutant enzymes containing a single tryptophan (L-Trp) residue. The fluorescence signatures of each of the three native L-Trp residues in PAH were determined by titrations with L-Phe. Trp187 primarily reports L-Phe induced allosteric conformational changes, while Trp120 reports active site L-Phe binding. Trp326 reports small signals of both active and allosteric site changes. Variable temperature stopped-flow fluorescence kinetic studies elucidated a working mechanism for L-Phe allosteric activation of PAH. Fluorescent signals from wild-type, single, and double L-Trp PAH mutants have been used to build kinetic mechanisms for the L-Phe binding in each subunit and subsequent active site reorganization or allosteric conformational change. In these mechanisms, the enzyme has reduced activity (1-2% of wtPAH) until both L-Phe induced active and allosteric site conformational changes have occurred. Failure of either activation step prevents enzyme turnover and is the chemical-based cause of the metabolic condition phenylketonuria.

The genetic disease cystic fibrosis is the most common lethal genetic disease in Western countries. People born with cystic fibrosis suffer from many health issues including severe respiratory problems, inflammation and recurrent lung infections that can become fatal. The disease is caused by the loss of function of a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an chloride ion channel and, in healthy people, its activity assures correct water and salt transport across the cell membrane. Most cases of cystic fibrosis are caused by a genetic defect that leads to the deletion of phenylalanine 508 (F508del) in the amino acid sequence of the protein. The molecular mechanism by which F508del leads to loss of function of the CFTR channel is still poorly understood. The mutation is found in the first nucleotide binding domain (NBD1) and studies have shown that it causes misfolding of CFTR and subsequent degradation of the protein by the cellular quality control system. It is established that the mutation affects stability and dynamics of NBD1 but does not alter its structure significantly. This destabilizing effect of F508del can be compensated by specific mutations distributed over different regions of NBD1, leading to recovery of membrane expression of a functional channel. A surprising example involves the regulatory insertion (RI), a 32-residue long segment found in all CFTR orthologs but not in related channels or transporters. The RI is not resolved in crystal structures of NBD1 nor cryo-EM structures of CFTR and has been described as intrinsically disordered. Its functional role in CFTR is unknown. Removal of the RI increases the stability of the NBD1 domain and, in the context of F508del-CFTR, this deletion restores maturation, cell surface expression and activity of the mutant channel. We probed the effect of the RI on NBD1 structure, dynamics and allostery using X-ray crystallography, single molecule FRET and hydrogen-deuterium exchange. We discovered that the RI enables an alternative NBD1 fold which departs markedly from the canonical fold previously observed for this domain and the NBDs of other ABC transporters. The conformational equilibrium between these states is regulated by ATP binding and affected by disease-associated conditions. Aside from clear alterations to structure and dynamics of NBD1, the RI also affects allostery, i.e. how NBD1 structure and dynamics respond to perturbations such as ligand binding. Finally, we show that the RI-enabled conformation is adopted in full-length CFTR and associated with increased channel activity in electrophysiological assays. We then identify an allosteric network that links the structural hotspots of the conformational changes to F508 and its surroundings. Lastly, we argue that these conformational changes lead to unfolding of NBD1 in the context of F508del, providing a new model for the molecular mechanism leading to pathogenesis. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Hsp70 molecular chaperones protect proteins from aggregation, assist in their native structure formation, and regulate stress responses in the cell. A mechanistic understanding of Hsp70 function will be necessary to explain its physiological roles and guide the therapeutic modulation of various disease states. To this end, several fundamental features of the Hsp70 structure-function relationship are investigated. The central component of Hsp70 chaperone function is its capacity for allosteric signaling between structural domains and tunable binding of misfolded protein substrates. In order to identify a cooperative network of sites that mediates interdomain allostery within Hsp70, a mutational correlation analysis is performed using genetic data. Evolutionarily correlations that describe an allosteric network are validated by examining roles for implicated sites in cellular fitness and molecular function. In a second component of the Hsp70 molecular mechanism, a novel function is discovered for the disordered C-terminal tail. This region of the protein enhances the refolding efficiency of substrate proteins independently of interdomain allostery and is required in the cell upon depletion of compensatory chaperones, suggesting a previously undescribed mode of chaperone action. Finally, experiments are initiated to assess the dynamic assembly of Hsp70 domains in various allosteric states and how domain orientations may be guided through interaction with partner co-chaperone proteins.

Cyclic nucleotides such as cAMP and cGMP serve as intracellular second messengers in diverse signaling pathways that control a wide range of cellular functions. Such pathways are regulated by key cyclic nucleotide receptor proteins including protein kinase A (PKA), the exchange protein directly activated by cAMP (EPAC), the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels, and protein kinase G (PKG), and malfunction of these proteins has been linked to a number of pathologies. While it is known that cyclic nucleotide binding to these proteins leads to structural perturbations that promote their activation, the role played by dynamics in auto-inhibition and cyclic-nucleotide-dependent activation is not fully understood. Therefore, in this thesis we examined dynamics within the cyclic-nucleotide receptor proteins EPAC, HCN and PKG, and found that dynamics are critical for allosteric control of activation and/or autoinhibition of all three proteins. In particular, our findings for EPAC and HCN have highlighted dynamics as a key modulator of the entropic and enthalpic components, respectively, of the free-energy landscape for cAMP-dependent allostery, while our findings for PKG have highlighted dynamics as a key determinant of the cGMP-vs.-cAMP selectivity necessary to minimize cross-talk between signaling pathways. Ultimately, we envision that the methods outlined in this thesis will reveal key differences in the regulatory mechanisms of human cyclic nucleotide receptors that can eventually be exploited in the development of novel therapeutics to selectively target a single receptor, and thus treat physiological conditions/diseases linked to malfunction of the target receptor. / Thesis / Doctor of Philosophy (PhD) / In this thesis, we examined cyclic-nucleotide-responsive proteins that regulate key physiological processes, and whose malfunction has been linked to cardiovascular and neurological disorders. In particular, in three such proteins we examined dynamics, whose role in cyclic-nucleotide-responsive function is not fully understood. We found that cyclic-nucleotide-dependent variations in dynamics play a critical role in the function of these proteins, with the results for each protein highlighting a different role played by dynamics. Ultimately, we envision that the methods outlined in this thesis will reveal key functional differences among human cyclic-nucleotide-responsive proteins that can eventually lead to the development of novel therapeutics to treat certain diseases such as arrhythmias or epilepsy by selectively targeting a single cyclic-nucleotide-responsive protein.

No description available.

Protein-ligand binding and protein allostery play a crucial role in cell signaling, cell regulation, and modern drug discovery. In recent years, experimental studies of protein structures including crystallography, NMR, and Cryo-EM are widely used to investigate the functional and inhibitory properties of a protein. On the one hand, structural classification and feature identification of the structures of protein kinases, HIV proteins, and other extensively studied proteins would have an increasingly important role in depicting the general figures of the conformational landscape of those proteins. On the other hand, free energy calculations which include the conformational and binding free energy calculation, which provides the thermodynamics basis of protein allostery and inhibitor binding, have proven its ability to guide new inhibitor discovery and protein functional studies. In this dissertation, I have used multiple different analysis and free energy methods to understand the significance of the conformational and binding free energy landscapes of protein kinases and other disease-related proteins and developed a novel alchemical-based free energy method, restrain free energy release (R-FEP-R) to overcome the difficulties in choosing appropriate collective variables and pathways in conformational free energy methods like umbrella sampling and metadynamics. / Chemistry