Academic literature on the topic 'Allosteric ligands'

AbstractProtein kinase A (cAMP dependent protein kinase catalytic subunit, EC 2.7.11.11) binds simultaneously ATP and a phosphorylatable peptide. These structurally dissimilar allosteric ligands influence the binding effectiveness of each other. The same situation is observed with substrate congeners, which reversibly inhibit the enzyme. In this review these allosteric effects are quantified using the interaction factor, which compares binding effectiveness of ligands with the free enzyme and the pre-loaded enzyme complex containing another ligand. This analysis revealed that the allosteric effect depends upon structure of the interacting ligands, and the principle “better binding: stronger allostery” observed can be formalized in terms of linear free-energy relationships, which point to similar mechanism of the allosteric interaction between the enzyme-bound substrates and/or inhibitor molecules. On the other hand, the type of effect is governed by ligand binding effectiveness and can be inverted from positive allostery to negative allostery if we move from effectively binding ligands to badly binding compounds. Thus the outcome of the allostery in this monomeric enzyme is the same as defined by classical theories for multimeric enzymes: making the enzyme response more efficient if appropriate ligands bind.

Allosteric modulators of G-protein-coupled receptors interact with binding sites that are topographically distinct from the orthosteric site recognized by the receptor's endogenous agonist. Allosteric ligands offer a number of advantages over orthosteric drugs, including the potential for greater receptor subtype selectivity and a more ‘physiological’ regulation of receptor activity. However, the manifestations of allosterism at G-protein-coupled receptors are quite varied, and significant challenges remain for the optimization of screening methods to ensure the routine detection and validation of allosteric ligands.

Allosteric communication within proteins is a hallmark of biochemical signaling, but the dynamic transmission pathways remain poorly characterized. We combined NMR spectroscopy and surface plasmon resonance to reveal these pathways and quantify their energetics in the glucocorticoid receptor, a transcriptional regulator controlling development, metabolism, and immune response. Our results delineate a dynamic communication network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, exhibits ligand- and coregulator-dependent dynamics coupled to graded activation. The allosteric free energy responds to variations in ligand structure: subtle changes gradually tune allostery while preserving the transmission pathway, whereas substitution of the entire pharmacophore leads to divergent allosteric control by apparently rewiring the communication network. Our results provide key insights that should aid in the design of mechanistically differentiated ligands.

In many proteins, especially allosteric proteins that communicate regulatory states from allosteric to active sites, structural deformations are functionally important. To understand these deformations, dynamical experiments are ideal but challenging. Using static structural information, although more limited than dynamical analysis, is much more accessible. Underused for protein analysis, strain is the natural quantity for studying local deformations. We calculate strain tensor fields for proteins deformed by ligands or thermal fluctuations using crystal and NMR structure ensembles. Strains—primarily shears—show deformations around binding sites. These deformations can be induced solely by ligand binding at distant allosteric sites. Shears reveal quasi-2D paths of mechanical coupling between allosteric and active sites that may constitute a widespread mechanism of allostery. We argue that strain—particularly shear—is the most appropriate quantity for analysis of local protein deformations. This analysis can reveal mechanical and biological properties of many proteins.

Abstract The structure of ligand-binding sites has been shown to profoundly influence the evolution of function in homomeric protein complexes. Complexes with multichain binding sites (MBSs) have more conserved quaternary structure, more similar binding sites and ligands between homologs, and evolve new functions slower than homomers with single-chain binding sites (SBSs). Here, using in silico analyses of protein dynamics, we investigate whether ligand-binding-site structure shapes allosteric signal transduction pathways, and whether the structural similarity of binding sites influences the evolution of allostery. Our analyses show that: 1) allostery is more frequent among MBS complexes than in SBS complexes, particularly in homomers; 2) in MBS homomers, semirigid communities and critical residues frequently connect interfaces and thus they are characterized by signal transduction pathways that cross protein–protein interfaces, whereas SBS homomers usually not; 3) ligand binding alters community structure differently in MBS and SBS homomers; and 4) except MBS homomers, allosteric proteins are more likely to have homologs with similar binding site than nonallosteric proteins, suggesting that binding site similarity is an important factor driving the evolution of allostery.

Allosteric antagonism by bitopic ligands, as reported for many receptors, is a distinct modulatory mechanism. Although several bitopic A2A adenosine receptor (A2AAR) ligand classes were reported as pharmacological tools, their receptor binding and functional antagonism patterns, i.e., allosteric or competitive, were not well characterized. Therefore, here we systematically characterized A2AAR binding and functional antagonism of two distinct antagonist chemical classes. i.e., fluorescent conjugates of xanthine amine congener (XAC) and SCH442416. Bitopic ligands were potent, weak, competitive or allosteric, based on the combination of pharmacophore, linker and fluorophore. Among antagonists tested, XAC, XAC245, XAC488, SCH442416, MRS7352 showed Ki binding values consistent with KB values from functional antagonism. Interestingly, MRS7396, XAC-X-BY630 (XAC630) and 5-(N,N-hexamethylene)amiloride (HMA) were 9–100 times weaker in displacing fluorescent MRS7416 binding than radioligand binding. XAC245, XAC630, MRS7396, MRS7416 and MRS7322 behaved as allosteric A2AAR antagonists, whereas XAC488 and MRS7395 antagonized competitively. Schild analysis showed antagonism slopes of 0.42 and 0.47 for MRS7396 and XAC630, respectively. Allosteric antagonists HMA and MRS7396 were more potent in displacing [3H]ZM241385 binding than MRS7416 binding. Sodium site D52N mutation increased and decreased affinity of HMA and MRS7396, respectively, suggesting possible preference for different A2AAR conformations. The allosteric binding properties of some bitopic ligands were rationalized and analyzed using the Hall two-state allosteric model. Thus, fluorophore tethering to an orthosteric ligand is not neutral pharmacologically and may confer unexpected properties to the conjugate.

Allostery is a form of protein regulation, where ligands that bind sites located apart from the active site can modify the activity of the protein. The molecular mechanisms of allostery have been extensively studied, because allosteric sites are less conserved than active sites, and drugs targeting them are more specific than drugs binding the active sites. Here we quantify the importance of allostery in genetic disease. We show that 1) known allosteric proteins are central in disease networks, contribute to genetic disease and comorbidities much more than non-allosteric proteins, and there is an association between being allosteric and involvement in disease; 2) they are enriched in many major disease types like hematopoietic diseases, cardiovascular diseases, cancers, diabetes, or diseases of the central nervous system; 3) variants from cancer genome-wide association studies are enriched near allosteric proteins, indicating their importance to polygenic traits; and 4) the importance of allosteric proteins in disease is due, at least partly, to their central positions in protein-protein interaction networks, and less due to their dynamical properties.

Historically, traditional screening for ligands has been optimized to detect standard orthosteric agonists and antagonists. However, with increasing emphasis on cellular functional screens, more allosteric ligands are being discovered as potential drugs. In addition, there are theoretical reasons (increased selectivity, better control of physiological systems, separate control of affinity and efficacy) allosteric ligands may be preferred therapeutic chemical targets. These factors may make it desirable to design high-throughput screens to specifically detect functionally allosteric ligands. This article discusses the unique features of allosteric ligands as drugs as well as the special conditions that should be considered to optimize a high-throughput screen toward the detection of allosteric drugs. Finally, the likelihood of detecting allosteric ligands that have direct effects on cells (either conventional agonism or functionally selective effects) is discussed as well as the optimization of detection of such ligands in screening assays.

Metabotropic glutamate receptors (mGluR) are members of the class C G-Protein Coupled Receptors (GPCR-s) and have eight subtypes. These receptors are responsible for a variety of functions in the central and peripheral nervous systems and their modulation has therapeutic utility in neurological and psychiatric disorders. It was previously established that selective orthosteric modulation of these receptors is challenging, and this stimulated the search for allosteric modulators. Fragment-Based Drug Discovery (FBDD) is a viable approach to find ligands binding at allosteric sites owing to their limited size and interactions. However, it was also observed that the structure-activity relationship of allosteric modulators is often sharp and inconsistent. This can be attributed to the characteristics of the allosteric binding site of mGluRs that is a water channel where ligand binding is accompanied with induced fit and interference with the water network, both playing a role in receptor activation. In this review, we summarize fragment-based drug discovery programs on mGluR allosteric modulators and their contribution identifying of new mGluR ligands with better activity and selectivity.

The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-mediated adenosine 3′,5′-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein–coupled receptor is not fully understood. Here, we show that extracellular Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor’s first extracellular loop as key determinants for Ca2+ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca2+ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTHR25C suggest that Ca2+ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.

Photopharmacology has the main purpose to allow the control of protein activity with light. The most exploited strategy used to achieve this objective is the freely diffusible photopharmacology and it is based in the use of photosensitive ligands. These ligands are small bioactive molecules, which include a part of their structure (i.e. photoswitch) that can experience molecular changes upon illumination with a determined wavelength of light. These ligands can freely diffuse and they can be applied with systems expressing native proteins. Azobenzene is the most common photoswitch used in photopharmacology and it can switch with near UV light from the flat and long trans isomer to a shorter bent cis configuration. The reverse photoisomerization can be achieved either with visible light or thermally with light. Thus, if we include azobenzene in the molecular scaffold of a ligand by means of a replacement of a particular moiety (azologization), we can obtain new azo compounds that will resemble to the original ligand, but their structural shape will dramatically changes upon illumination (photoisomerization). Therefore, the two possible isomers will have distinct binding modes to the target protein and will lead to different protein activities under different light conditions, which is known as photoswitching. Metabotropic Glutamate Receptors (mGluRs) belong to the class C/Glutamate family of G Protein-Coupled Receptors and control many neuronal and glial functions. mGlu receptors are endogenously activated by glutamate, which is the major excitatory neurotransmitter in the central nervous system (CNS), but they can also be activated or inactivated by allosteric modulators. They are usually considered better drug candidates than the orthosteric ligands because usually highly specific for a receptor and able to modulate the activity of a given receptor without blocking endogenous ligand binding. First of all, we designed and synthesized three families of compounds, using an azo-replacement strategy, to obtain photoswitchable allosteric modulators with possible NAM activity in mGlu5 in the cis isomers, while in the trans form they are inactive. This behavior is easily controlled by illuminating with different wavelengths and it is reversible in vitro. All the three families were inactive as NAMs, but some results suggest that the compounds could act as mGlu5 PAMs in trans form. Studies are continuing in this direction (Chapter 1). Next, we carry out the design and synthesis of compounds to improve PAM activity at the mGlu4 receptor and increase selectivity over the other group III mGluRs of at least one azo benzene candidate with a structure similar to Optogluram, the first photoswitchable positive allosteric modulator for the mGlu4 receptor. We obtained Optogluram-2 with good pharmacological potency and improved the photoisomerization properties. Under 380 nm light, the potency of Optogluram-2 is significantly reduced. The change in photoinduced potency observed is greater in Optogluram-2 than in Optogluram. Optogluram-2 has similar potency to Optogluram but is more selective for mGlu4 both on the receptors of the same group III as on the other mGluRs. All this indicates that Optogluram-2 can induce an improved activated/deactivated profile change as well as have an optimal selectivity for more complex assays, such as in vivo assays (Chapter 2). Additionally, we synthesized two series of compounds to find the first photoswitchable compound to selectively enable optical control of the endogenous mGlu1 receptor. Photoglurax-1 arose as a PAM of mGlu1 with micromolar potency in the trans isomer. Under 380nm light, the potency is significantly reduced. Photoglurax-1 turned out to be an equipotent mGlu4 PAM and therefore its general profile is not suitable for in vivo translation as a possible mGlu1 PAM tool compound. However, a dual mGlu1/mGlu4 PAM activity could be intriguing for an antipsychotic agent, since mGlu4 PAM activity can alleviate catalepsy, a major adverse event with standard antipsychotic drug treatment. In contrast, Photoglurax-2 acts as a mGlu1 PAM and does not show any observable allosteric effect on mGlu4 or activity on mGlu5, and therefore Photoglurax-2 represents a potential in vivo photoswitchable PAM mGlu1 tool compound. Reversible monitoring of mGlu1 activity obtained with light can be very advantageous in studying the pharmacological and physiological implications of mGlu1 in many diseases with unprecedented precision (Chapter 3). Finally, we designed and synthesized a family of novel photoswitchable azoheteroarenes as mGlu1 NAMs with an active trans isomer and an inactive cis isomer to reversibly inactivate the function of the mGlu1 receptor. The potencies of the trans configurations of some compounds of the family are in the micromolar range . Unfortunately, after 400 nm illumination the results were inconclusive due to artifacts that could originate from a possible toxicity of cis azo compounds. More experiments should be done with cells that do not express mGlu1 and also changing the light system to corroborate eventual toxicity (Chapter 4). Likewise, we use some of these compounds in their trans form, therefore without applying light, as tools to expand the knowledge about the nature of the intermediate states induced by mGlu receptor agonists in studies of fluorescence conformational dynamics. Analysis of the effect of mGlu1 NAMs on receptor conformational changes is reported in Chapter 4.
Los receptores metabotrópicos de glutamato (mGlu) son GPCRs distribuidos a través del CNS y se consideran dianas farmacológicas para trastornos neurológicos, tales como el dolor neuropático y la enfermedad de Parkison, entre otras. En primar lugar, diseñamos y sintetizamos tres familias de compuestos, utilizando una estrategia de azo- reemplazo, para obtener moduladores alostéricos de GPCR fotoconmutable con posible actividad NAM en mGlu5 en los isomeros cis, mientras que en la disposición trans son inactivos. Este comportamiento se controla fácilmente con iluminación con diferentes longitudes de onda y es reversible in vitro. Ninguna familia resultò activa como NAMs, pero algunos resultados sugieren que los compuestos podrían actuar como PAMs mGlu5 en forma trans. La investigación continúa siguiendo esta dirección (Capítulo 1). Seguidamente, realizamos el diseño y sintesis de compuestos para mejorar la actividad de PAM en el receptor mGlu4 y aumentar la selectividad sobre los otros mGluR del grupo III de al menos un candidato a azobenceno con estructura similar a Optogluram, el primer modulador alostérico positivo fotoconmutable para el receptor mGlu4. Obtuvimos Optogluram-2 con buena potencia farmacologica y mejoramos las propriedades de fotoisomerizacion. Bajo una luz de 380 nm, la potencia de Optogluram-2 se reduce significativamente. El cambio de potencia fotoinducido observado es mayor en Optogluram-2 que en Optogluram.Optogluram-2 tiene potencia parecida a Optogluram pero es màs selectivo para mGlu4 tanto sobre los receptores del mismo grupo III como sobre los demas. Todo esto indica que Optogluram-2 puede inducir un cambio de perfil activado/desactivado mejorado asì como tener una selectividad optimal para ensayos más complejos, como los ensayos in vivo (Capítulo 2). Sintetizamos dos series para encontrar el primer compuesto fotoconmutable para habilitar selectivamente el control óptico del receptor mGlu1 endógeno. Photoglurax-1 surgió como un PAM de mGlu1 con potencia micromolar en el isómero trans. Bajo una luz de 380 nm, la potencia se reduce significativamente. Photoglurax- 1 resultó ser un mGlu4 PAM equipotente y por eso su perfil general no es apropiado para una traducción in vivo como una posible herramienta molecular mGlu1 PAM. Sin embargo, una actividad dual mGlu1/mGlu4 PAM podría ser intrigante para un agente antipsicótico,ya que la actividad mGlu4 PAM puede aliviar la catalepsia, un evento adverso importante con el tratamiento estándar con fármacos antipsicóticos. En cambio, Photoglurax-2 actúa como un PAM mGlu1 y no muestra ningún efecto alostérico observable en mGlu4 ni actividad en mGlu5 y por lo tanto Photoglurax-2 representa una potencial herramienta molecular PAM mGlu1 fotoconmutable in vivo. El control reversible de la actividad de mGlu1 obtenido con luz puede ser muy ventajoso para estudiar las implicaciones farmacológicas y fisiológicas de mGlu1 en muchas enfermedades con una precisión sin precedentes (Capítulo 3). Finalmente, intentamos diseñar y sintetizar una familia de novedosos azoheteroarenos fotoconmutables como NAMs de mGlu1 con un isomero trans activo y un isomero cis inactivo para inactivar reversiblemente la función del receptor mGlu1. Las potencias de las configuraciones trans de algunos compuestos de la familia estan en el rango de micromolaridad. Desafortunadamente, tras una iluminación de 400 nm los resultados fueron no concluyentes debido a artefactos que podrían originarse a partir de una posible toxicidad de los compuestos cis azo. Se deben realizar más experimentos con células que no expresen mGlu1 y cambiando tambien el sistema de luz para comprobar si se trata de toxicidad (Capítulo 4). Asimismo, utilizamos algunos de estos compuestos en su forma trans, por lo tanto sin aplicar luz, como herramientas para ampliar el conocimiento sobre la naturaleza de los estados intermedios inducidos por agonistas de los receptores mGlu en estudios de dinámica conformacional de fluorescencia. El análisis del efecto de los NAMs de mGlu1 sobre los cambios conformacionales del receptor están reportados en el Capítulo 4. En resumen, encontramos como obtener un interruptor molecular entre varias actividades farmacologicas. Ademàs, demostramos que la fotofarmacologia presenta ventajas respecto a la farmacologia convencional, ya que permite ajustar la activacion del receptor con luz.

In the brain events happen in the scale of milliseconds, and the fine processes of neurons and neuroglia are highly compartmentalized at a microscopic level. These exclusive features of the brain define extremely precise temporal and spatial patterns of cellular activity, which are of fundamental importance for its proper functioning, because they allow the fast processing, sorting, integration, and flow of information with high reproducibility and precision. To gain deeper understanding of how these patterns are organized in time and space, we need new tools that overcome the spatiotemporal limitations of the currently available techniques. Recently, neurobiology was revolutionized by the idea of using light to control neuronal proteins remotely with millisecond- and micrometer-precision, which led to the development of a new field of study called optogenetics. After more than a decade, the control of protein function with light has gone far beyond optogenetics and its need of genetic manipulations. Optopharmacology is now gaining importance because it is less invasive and suitable for controlling endogenous proteins with light, and each year compounds with enhanced photochemical and pharmacological properties are developed. This thesis reviews the optopharmacological tools developed so far to study a family of neuronal proteins called metabotropic glutamate (mGlu) receptors. We are interested in these proteins because they participate in neurotransmission, and are link to neuropathologies when dysfunctional. Thanks to pioneering advances in probing these receptors with light, many features of mGlu signaling have been unraveled, and it is now emerging that these receptors follow activation mechanisms different from those initially foreseen. Still, it is not clear what their exact kinetics are, or which are the functional consequences of temporal and spatial patterns of activity – which are widespread both among brain structures and across evolution. Despite the fundamental relevance of mGlu receptors to brain computing in physiology and disease, the mechanisms that govern their functioning are still partially understood, and this is mainly due to the scarcity of tools to activate mGlu receptors with spatiotemporal precision. The aim of this thesis was to expand the toolbox of optical switches to activate with light mGlu receptors, with special interest in respecting the physiological context of their activation. For that purpose, we discarded approaches based on genetic engineering of receptors, as well as irreversible uncaging of compounds. We preferred the use of optopharmacology, and specifically applied it to allosteric modulators, which display higher selectivity and more physiological activation than orthosteric ligands. This objective implied technical challenges due to the structural restrictions of mGlu allosteric binding pockets, but at the same time offered high gains to spatiotemporally control these receptors both in therapeutic and basic research applications. From these premises, we: 1. developed the first light-regulated allosteric modulators targeting metabotropic glutamate receptors. The molecular design, in vitro and in vivo characterization of alloswitch-1 and G4optoNAM are presented in Chapters 1-2. 2. expanded the knowledge about this new class of compounds through a library of compounds derived from alloswitch-1, and present the inferred data about structure-activity relationship and optimal optopharmacological characteristics for allosteric photoswitches of mGlu receptors (Chapter 3). 3. demonstrated the functional photoisomerization of alloswitch-1 by using two-photon stimulation, with the aim of exploring the resolution limits of reversible optical switches (Chapter 4). Overall, this thesis shows for the first time the design and characterization of optical switches for the allosteric and remote control of endogenous mGlu receptors in vitro and in vivo with light. This advance broadens the availability of optical tools in research to manipulate mGlu receptors with high temporal and spatial resolution, and represents a step forward in innovative opportunities to treat neuropathologies with light.
En el sistema nerviós els esdeveniments es desenvolupen en l’escala temporal dels milisegons, i els processos que tenen lloc en neurones i cèl·lules de la glia presenten compartimentalitzacions microscòpiques. Aquesta organització determina uns patrons d’activitat ben definits temporal i espacialment, els quals permeten el precís funcionament del sistema nerviós per tal de transmetre, integrar i processar la informació d’una forma rapida i especifica. Per entendre millor el modus operandi del cervell en el temps i l’espai, calen noves eines que permetin superar les limitacions espaitemporals de les tecnologies existents per l’observació passiva o l’activa manipulació del sistema nerviós. Una de les estratègies més rapides i precises per activar e inactivar proteïnes neuronals es basa en la seva fotosensibilització, per tal de poder-les controlar mitjançant la precisió espai-temporal incomparable que la llum ofereix. Aquesta tesi fa un resum de les eines òptiques disponibles per detectar (sensors) e induir (commutadors) l’activitat d’una família de proteïnes neuronals denominades receptors metabotropics de glutamat (mGlu). Estem interessats en aquests receptors per la importància que tenen com moduladors de la neurotransmissió, i el seu rol en el desenvolupament de neuropatologies. L’objectius de la tesi fou desenvolupar eines optofarmacològiques pel control òptic i reversible dels receptors mGlu amb llum, considerant els grans avantatges d’especificitat espaitemporal que ofereix el fotocontrol de proteïnes i l’escassetat de tals eines. El primer capítol descriu el disseny, la síntesi i la caracterització d’alloswitch-1, el primer fotocommutador al·lostèric capaç d’activar receptors mGlu amb llum de forma reversible. El segon capítol il·lustra la caracterització de G4optoNAM, un fotocommutador al·lostèric actiu en receptors mGlu4. El tercer capítol recull una llibreria de compostos derivats del precursor alloswitch-1 amb diverses substitucions químiques, que presenten característiques fotofísiques i optofarmacològiques variades. Al quart i últim capítol demostrem la capacitat dels alloswitches de fotoisomeritzar amb il·luminació micromètrica amb un làser multifotó. La nostra capacitat d’expandir el ventall d’eines optofarmacològiques que permeten un control farmacològic de receptors neuronals amb llum, de forma remota i no invasiva, ha aportat a la comunitat científica noves metodologies farmacològiques per a l’estudi de la fisiopatologia del sistema nerviós.

Le récepteur du glucagon-like peptide-1 (GLP-1) (GLP-1R) est un récepteur couplé aux protéines G de classe B et une cible médicamenteuse importante dans le traitement du diabète de type 2 (DT2)
The glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) is a class B G protein-coupled receptor and an important drug target in the treatment of type 2 diabetes (T2D)

Molecular recognition, defined as the specific interactions between two or more molecules, is at the center of many biological processes including catalysis, signal transduction, gene regulation and allostery. Allosteric regulation is the modification of function caused by an intermolecular interaction. Allosteric proteins modify their activity in response to a biological signal that is often transmitted through the interaction with a small effector molecule. Therefore, determination of the origins of intermolecular interactions involved in molecular recognition and allostery are essential for understanding biological processes. Classically, molecular recognition and allosteric regulation have been associated to structural changes of the system. NMR spectroscopic methods have indicated that changes in protein dynamics may also contribute to molecular recognition and allostery. This thesis is an investigation of the contributions of both structure and dynamics in molecular binding phenomena. In chapter I, I describe molecular recognition, allostery and examples of allostery and cooperativity. Then I discuss the contribution of protein dynamics to function with a special focus on allosteric regulation. Lastly I introduce the hemoglobin homodimer, HbI of Scapharca inaequivalvis and the mRNA binding protein TIS11d. Chapter II is the primary focus of this thesis on the contribution of protein dynamics to allostery in the dimeric hemoglobin of scapharca inaequivalvis, HbI. Thereafter I concentrate on the mechanism of adenine recognition of the Tristetraprolin-like (TTP) protein TIS11d; this study is detailed in Chapter III. In Chapter IV I discuss broader impacts and future directions of my research. This thesis presents an example of the use of protein NMR spectroscopy to probe ligand binding. The studies presented in this thesis emphasize the importance of dynamics in understanding protein function. Measurements of protein motions will be an element of future studies to understand protein function in health and disease.

The Trk family of tyrosine kinase receptors and the common p75NTR receptor are neurotrophin receptors. Nerve growth factor (NGF) binds TrkA, brain-derived neurotrophic factor (BDNF) binds TrkB, and neurotrophin-3 (NT-3) binds TrkC. The extracellular domain of the Trk receptor has five subdomains: a leucine-rich motif (D2), two cysteine-rich motifs (D1, D3) and immunoglobulin-like subdomains Ig-C1 (D4) and Ig-C2(D5). The Trk D4 subdomain regulates ligand-independent activation. The TrkA-D5 and TrkB-D5 subdomains regulate cognate ligand binding and Trk activation. However, the p75NTR receptor binds all neurotrophins and regulates ligand affinity and Trk signals. We showed that p75NTR affects Trk ligand - binding and activation of Trks by changing Trk subdomain utilization. When p75NTR is coexpressed, NGF can activate TrkA via the cysteine-1 subdomain (D1), and BDNF can activate TrkB via leucine-rich motif (D2) and cysteine-2 (D3) subdomains. We hypothesized conformational or allosteric regulatory mechanisms. To further study the interactions between ligands and Trks, we examined TrkA binding to NT-3 as a heterologous ligand because these interactions are biologically relevant. We found the TrkA “hot spot” functional docking sites used by NT-3. We demonstrate that TrkA-D5 has partially overlapping but distinct binding and activation “hot spots” for both, NGF and NT-3. Moreover, ligand - binding studies have identified additional NT-3 binding/allosteric site on TrkA-D4. NT-3 binding to both sites induces full agonism. Conversely, the TrkA-D5 NT-3 binding site is partially agonistic, but antagonizes NGF activity. Lasly, we address NT-3 binding and activation sites on the TrkC receptor by raising a monoclonal antibody that recognizes the juxtamembrane-linker domain of the TrkC receptor. This antibody is an artificial TrkC receptor agonist. The epitope of mAb 2B7 defines a previously unknown hot spot of TrkC. Binding to this “hot spot” induces survival but n
La famille de récepteurs de Trk tyrosine kinase et le récepteur p75NTR sont des récepteurs de neurotrophines. Le facteur de croissance nerveuse (NGF) intéragit avec le récepteur TrkA, le facteur neurotrophique dérivé du cerveau (BDNF) intéragit avec le récepteur TrkB et la neurotrophine-3 (NT-3) intéragit avec TrkC. Le domaine extracellulaire du récepteur Trk contient cinq sous-domaines: un motif riche en leucine (D2), deux motifs riches en cysteine (D1, D3) et des sous-domaines de type immunoglobuline Ig-C1(D4) et Ig-C2(D5). Le sous-domaine Trk D4 régule l'activation indépendante de ligand. Les sous-domaines TrkA-D5 et TrkB-D5 régulent la liaison de ligands endogènes ainsi que l'activation du récepteur Trk. Le récepteur p75NTR intéragit avec toutes les neurotrophines et régule l'affinité des ligands et les signaux issues de l'activation du récepteur Trk. Par ailleurs, nous avons démontré que le p75NTR affecte la liaison du ligand au récepteur Trk en changeant l'activation de l'utilisation des sous-domaines. Lorsque le recepteur de p75NTR est coexprimé, le NGF peut activer le récepteur TrkA via le sous-domaine cysteine-1 (D1) et BDNF peut activer TrkB via le motif riche en leucine (D2) ainsi que via le sous-domaine cysteine-2 (D3). Nous avons examiné la liaison d'un ligand hétérologue, NT-3 sur le récepteur TrkA afin d'étudier plus profondément les interactions entre les ligands et le récepteur TrkA. Ces interactions sont biologiquement pertinentes. Pour faire ceci, nous avons tout d'abord identifié les « points chauds » présents sur le récepteur TrkA qui servent des sites d'amarrage fonctionnels du ligand NT-3. Nous avons démontré que le sous domaine TrkA-D5 possède deux points chauds distincts, notamment un point chaud qui sert comme le site d'amarrage et d'activation du NGF et un point chaud qui sert comme le site d'amarrage et d'activation de la NT-3. Toutefois, ces deux sites d'amarrage se chevauchent partiellement. D

Downstream regulatory antagonist modulator (DREAM) is a calcium sensing protein that co-assembles with KV4 potassium channels to regulate ion currents as well as with DNA in the nucleus, where it regulates gene expression. The interaction of DREAM with A-type KV4 channels and DNA has been shown to regulate neuronal signaling, pain sensing, and memory retention. The role of DREAM in modulation of pain, onset of Alzheimer’s disease, and cardiac pacemaking has set this protein as a novel therapeutic target. Moreover, previous results have shown a Ca2+ dependent interaction between DREAM and KV4/DNA involving surface contacts at the N-terminus of DREAM. However, the mechanisms by which Ca2+ binding at the C-terminus of DREAM induces structural changes at the C- and N-terminus remain unknown. Here, we present the use of biophysics and biochemistry techniques in order to map the interactions of DREAM and numerous small synthetic ligands as well as KV channels. We further demonstrate that a highly conserved network of aromatic residues spanning the C- and N-terminus domains control protein dynamics and the pathways of signal transduction on DREAM. Using molecular dynamics simulations, site directed mutagenesis, and fluorescence spectroscopy we provide strong evidence in support of a highly dynamic mechanism of signal transduction and regulation. A set of aromatic amino acids including Trp169, Phe171, Tyr174, Phe218, Phe235, Phe219, and Phe252 are identified to form a dynamic network involved in propagation of Ca2+ induced structural changes. These amino acids form a hydrophobic network connecting the N- and C-terminus domains of DREAM and are well conserved in other neuronal calcium sensors. In addition, we show evidence in support of a mechanism in which Ca2+ signals are propagated towards the N-terminus and ultimately lead to the rearrangement of the inactive EF-hand 1. The observed structural motions provide a novel mechanism involved in control of the calcium dependent KV4 and DNA binding. Altogether, we provide the first mechanism of intramolecular and intermolecular signal transduction in a Ca2+ binding protein of the neuronal calcium sensor family.

The known functional ionotropic glutamate receptors (iGluRs) are composed of three major subtypes: AMPA, NMDA, and kainate. In 1998, in the landmark paper discussed in this chapter, Armstrong et al. provided the first crystal structure of an iGluR-subunit ligand-binding core, the S1S2 region of the rat GluA2 ‘flop’ isoform. They solved its structure with X-ray crystallography from selenomethonine crystals. They also identified residues involved in kainate binding, analysed allosteric sites that regulate affinity and specificity of the agonist, and mapped potential subunit–subunit interaction sites. They also proposed that binding of different agonists may result in variable degrees of domain closure. This work has profound impact on the field and it has been importantly cited. Subsequently, numerous high-resolution crystal structures of ligand-binding domains of iGluRs in complex with ligands, both agonists and antagonists, have been solved.

Models and methods of statistical thermodynamics and physical adsorption theory allow us to quantitate cooperative interactions between ligand molecules adsorbed on a macromol-ecule, in particular allosteric effects described in detail in oxygen binding to hemoglobin. We show how the Hill equation can be modified and how the Hill coefficient can be interpreted. On the other hand, the entropy of the adsorption system allows us to visualize cooperative binding processes. Cooperative interactions lead to a decrease in the entropy of the system and an increase in the information it contains.