Nurr1 is a transcription factor in the nuclear receptor family that is responsible for midbrain dopaminergic neuron development and maintenance. Unlike traditional nuclear receptor signaling regulation, Nurr1 activity is not modulated by a ligand or by coregulator protein binding to its activation function-2 site. Several mechanisms for Nurr1 regulation have been proposed, including interaction with other signaling pathways. The Wnt/beta-Catenin pathway is important for proper development of many tissues, including the midbrain. Recently it has been shown that activation of this pathway stimulates the activity of Nurr1 through the direct interaction of Nurr1 with beta-catenin. Therefore, in this study I set out to define the biophysical details of this interaction. My results show that the use of expressed domains of these proteins will be useful to further examine the Nurr1/beta-catenin interaction through crystallography and cross-linking.

Clathrin mediated endocytosis is a fundamental cellular process used to regulate the response of cells to their environment. It has been usurped as a means for pathogens to enter cells. Clathrin mediated endocytosis is driven by the polymerization of clathrin into a polyhedral lattice. Lattice formation is critical for stabilization of membranes and organization of the protein machinery used to form a budding vesicle. Clathrin has a unique three legged structure. The legs interweave to form the lattice. Regulation of the process of lattice assembly has been observationally described for over twenty years. The mechanisms of regulation are only now being defined.

The clathrin endocytic machinery is closely associated with the actin cytoskeleton. This may allow the force of actin polymerization to be harnessed for coated vesicle formation or actin may play an additional stabilizing role during endocytosis. The interaction between clathrin and actin machineries is becoming an increasingly important research area. The details of the link between clathrin and actin are not yet determined.

To help define the mechanisms involved in forming clathrin coated vesicles and linking clathrin to the actin cytoskeleton biochemical and structural studies were undertaken. The first structure of the regulatory clathrin light chain suggested a conformational switch regulated clathrin assembly. Structural and biochemical work on the huntingtin interacting proteins again suggested a conformational switch regulates the link between clathrin and actin.

This work suggests a general mechanism of numerous conformational switches being flipped to regulated clathrin mediated endocytosis.

Every signaling event in biology begins with a molecular recognition process. In order for information to directly pass between two molecules, they must come in contact with each other for the requisite time to convey a given message. Specialized pathways often involve cascades of high fidelity interactions, near deterministically arriving at the same outcome for a given input. Other signals are capable of exerting disparate effects depending on the cellular context. Promiscuity in molecular recognition plays a central role in the capacity to produce different responses to the same signal.

For the steroid hormone receptors, the input signal is a small molecule ligand. Upon binding of the hormone, these molecules move to the nucleus, localize to different genomic response elements, and regulate the transcription levels of many different genes by recruitment of transcriptional machinery. The abilities to recognize multiple DNA sequences and to recruit different coregulators require that these interaction surfaces adapt to and engage with diverse structures over a relatively narrow spectrum of interaction energies.

The essential functions of DNA recognition and coregulator binding were investigated using dynamic structural analyses and biophysical measurements of interaction energies. For both, the manifestations of structural remodeling and energetic perturbation at sites removed from the interaction surfaces serve as evidence for allosteric regulation. In DNA recognition, we learn that nucleotide sequence modulates the structure of the dimerization surface of the glucocorticoid receptor, affecting both the cooperativity of complex formation and the kinetics of dissociation. In coregulator binding to the androgen receptor, we find distant structural remodeling and energetic coupling to a site of post translational modification. Together, these allosteric mechanisms help explain functional discrimination in a background of energetic degeneracy.

Steroid receptors are regulated by a diverse array of cellular effectors. Interactions with chaperone proteins, steroid hormones, kinase cascades, motor proteins, co-regulator proteins, and DNA determine the functional output of a receptor. Co-regulator proteins directly bind SRs at a surface site on the C-terminal ligand binding domain, apart from the buried ligand binding pocket, and are capable of activating and repressing transcriptional activity through the receptor. Most SRs prefer to bind co-regulators bearing the NR box motif LXXLL, where X is any amino acid. The sequences surrounding the Leu residues and the amino acids that comprise the receptor's activation function-2 surface determine the specificity of the interaction. The recruitment patterns of co-regulator proteins to SRs is key in determining cell and tissue-specific responses to natural and synthetic therapeutic steroids.

Androgen receptor is unlike the other SRs in that it preferentially binds co-regulators with aromatic residues comprising the NR box. To investigate this unusual preference, we solved X-ray structures of several aromatic-containing peptides bound to AR's AF-2 surface, and determined binding constants for the interactions by surface plasmon resonance (Chapter 2). Several AR residues were shown to be involved in an induced-fit mechanism that allows the receptor to recognize a wider variety of motifs.

Co-regulator recruitment to GR's AF-2 surface was investigated biochemically, revealing that binding events at AF-2 and the ligand binding pocket are allosterically coupled (Chapter 1). We found that NR box binding to GR's AF-2 slows association and dissociation of a fluorescent GR agonist, using fluorescence polarization. The effect on steroid binding was NR box sequence dependent with 7 of 18 peptides assayed showing the ability to reduce steroid binding rates significantly. Dissociation constants were determined for 8 of 18 peptides, revealing a more complete consensus sequence for NR box binding to GR. Additionally, mutation of a residue conserved in SRs shown to effect chaperone, steroid, and co-regulator binding to GR in cells was confirmed as a key component of the network connecting steroid and co-regulator binding sites.

Numerous small molecules serve to coordinate many biological activities within cells of multicelluar organisms. These ligand-receptor interactions are vital to the control of cellular processes such as altering cellular membrane potential and activation of gene transcription. This dissertation includes two ligand-receptor interactions projects: validating a small molecule as a selective photoreactive antagonist, ANQX, for AMPA receptors at synapses using x-ray crystallography and mass spectroscopy; and, utilizing a directed evolution yeast based selection assay to re-engineer the androgen receptor to activate transcription with gestrinone, a proof-of-concept drug. These two projects combined provide additional insight into the molecular basis of ligand-receptor interactions of ion channels and steroid nuclear receptors.

The thyroid hormone receptor is a ligand-dependent transcription factor crucial for the regulation of a wide variety of processes involved in metabolism, differentiation, and development. While much has been learned about the structure of the thyroid hormone receptor and the molecular determinants of hormone binding, a complete understanding of these determinants has not yet been obtained.

In order to better understand the molecular determinants of hormone binding in the thyroid hormone receptor, v-erbA, a naturally occurring form of the receptor found in a retrovirus, was used as a platform for mutational studies. The use of v-erbA, which is severely compromised in its ability to bind thyroid hormone and activate transcription, provided a convenient and natural template upon which targeted mutations could be made.

Mutational analysis on ligand binding domain constructs determined that the addition of a missing helix (Helix 12) near the C-terminus of v-erbA was insufficient to restore ligand binding ability to the receptor, but an additional single point mutation, S395F, near the same location was capable of restoring hormone affinity to within ten-fold of that of wild-type thyroid hormone receptor. Protein stability assays based on protease degradation and thermal denaturation also demonstrated this constructs improved stability over other v-erbA constructs.

Transcriptional data obtained using full-length constructs in transient transfection assays demonstrated that the S395F mutation significantly enhanced the ability of v-erbA to activate transcription in the presence of hormone. While both v-erbA and v-erbA + Helix 12 constructs not only failed to activate transcription in the presence of hormone but maintained repression seen in the absence of hormone, the v-erbA + Helix 12 S395F mutant activated transcription even beyond the level seen with wild-type thryroid hormone receptor.