Crystal structure of the ZP-N domain of ZP3 reveals the core fold of animal egg coats

Species-specific recognition between the egg extracellular matrix (zona pellucida) and sperm is the first, crucial step of mammalian fertilization. Zona pellucida filament components ZP3 and ZP2 act as sperm receptors, and mice lacking either of the corresponding genes produce oocytes without a zona pellucida and are completely infertile. Like their counterparts in the vitelline envelope of non-mammalian eggs and many other secreted eukaryotic proteins, zona pellucida subunits polymerize using a 'zona pellucida (ZP) domain' module, whose conserved amino-terminal part (ZP-N) was suggested to constitute a domain of its own. No atomic structure has been reported for ZP domain proteins, and there is no structural information on any conserved vertebrate protein that is essential for fertilization and directly involved in egg-sperm binding. Here we describe the 2.3 ångström (A) resolution structure of the ZP-N fragment of mouse primary sperm receptor ZP3. The ZP-N fold defines a new immunoglobulin superfamily subtype with a beta-sheet extension characterized by an E' strand and an invariant tyrosine residue implicated in polymerization. The structure strongly supports the presence of ZP-N repeats within the N-terminal region of ZP2 and other vertebrate zona pellucida/vitelline envelope proteins, with implications for overall egg coat architecture, the post-fertilization block to polyspermy and speciation. Moreover, it provides an important framework for understanding human diseases caused by mutations in ZP domain proteins and developing new methods of non-hormonal contraception

Molecular Dynamic Studies of Nuclear Receptors Ligand Binding Domain

Nuclear Receptors (NR) function as transcription factors that regulate genes that affect processes like reproduction, development and metabolism. The NRs are activated upon a given signal, which can be a ligand binding or a chemical modification. When activated, the receptors perform a conformational change that opens up interaction surfaces on the ligand binding domain, where coactivators can bind and transcription of the target gene can start. In this thesis the key events for NR activation, the ligand binding/unbinding mechanism and interactions with cofactors, are studied. Furthermore the communication between the ligand binding pocket and the cofactor interaction surface was investigated. To gain insight on the ligand unbinding mechanism we performed molecular dynamics (MD) studies on two NRs. The unbinding mechanism from Retinoic Acid Receptor (RAR) and Estrogen Receptor (ER) was studied with modified MD methods, random acceleration MD and steered MD. In the RAR study 4 unbinding pathways of the ligand retinoic acid were obtained, where one of the pathways were more likely than the others. Thus ligand binding could be obtained without major conformational changes on the receptor structure. In the ER study, three different ligands unbinding from ER α and β was studied. The results showed that an ER agonist or selective agonist could unbind from the receptor without causing major conformational changes, while a slightly more bulky antagonist could not. Thus NR agonist and antagonist would use different unbinding mechanisms. The results from the ER simulations also showed variance in pathway preference between the different ligands. Differences between the ligands and receptor subtypes might therefore also effect the unbinding and hence influence ligand selectivity. When the NR is activated an interaction surface becomes available and cofactors with a conserved motif can bind. In MD studies of Liver Receptor Homologue 1 (LRH-1) and Liver X Receptor (LXR), the interaction between different cofactor peptides and the receptors were characterized. In the LRH-1 study, a specific interaction from an aspartate to the motif was identified while the interactions between LXR and cofactor peptides showed a less specific binding. Thus specificity between LXR-cofactors should be found in other factors. Cofactor interactions were also studied in the context of ligand binding. For LRH-1, a bound ligand to the receptor caused different effects on the receptor- cofactor peptides interaction. This indicates a communication pathway between the ligand and the cofactor peptide, an allosteric communication. Allosteric signaling is difficult to study, to do so we used a modified MD technique, anisotropic thermal diffusion method. With this method we were able to identify an allosteric signaling pathway from a coactivator peptide through LXR to the ligand in the ligand binding pocket

Small molecule binding to proteins: affinity and binding/unbinding dynamics from atomistic simulations

A dynamic situation! Molecular dynamics simulations at equilibrium are shown to correctly identify the binding mode of dimethyl sulfoxide in the rotamase FKBP without using any experimental information or bias. Furthermore, both the binding and unbinding kinetics were found to have a double-exponential time dependence

Exploring the molecular basis of action of ring D aromatic steroidal antiestrogens

Salpichrolides are natural plant steroids that contain an unusual six-membered aromatic ring D. We recently reported that some of these compounds, and certain analogs with a simplified side chain, exhibited antagonist effects toward the human estrogen receptor (ER), a nuclear receptor whose endogenous ligand has an aromatic A ring (estradiol). Drugs acting through the inhibition or modulation of ERs are frequently used as a hormonal therapy for ER(+) breast cancer. Previous results suggested that the aromatic D ring was a key structural motif for the observed activity; thus, this modified steroid nucleus may provide a new scaffold for the design of novel antiestrogens. Using molecular dynamics (MD) simulation we have modeled the binding mode of the natural salpichrolide A and a synthetic analog with an aromatic D ring within the ERα. These results taken together with the calculated energetic contributions associated to the different ligand-binding modes are consistent with a preferred inverted orientation of the steroids in the ligand-binding pocket with the aromatic ring D occupying a position similar to that observed for the A ring of estradiol. Major changes in both dynamical behavior and global positioning of H11 caused by the loss of the ligand–His524 interaction might explain, at least in part, the molecular basis of the antagonism exhibited by these compounds. Using steered MD we also found a putative unbinding pathway for the steroidal ligands through a cavity formed by residues in H3, H7, and H11, which requires only minor changes in the overall receptor conformation.Fil: Alvarez, Lautaro Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Veleiro, Adriana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; ArgentinaFil: Burton, Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentin

Unbinding of Retinoic Acid from the Retinoic Acid Receptor by Random Expulsion Molecular Dynamics

Unbinding pathways of retinoic acid (RA) bound to retinoic acid receptor (RAR) have been explored by the random expulsion molecular dynamics (REMD) method. Our results show that RA may exit the binding site of RAR through flexible regions close to the H1-H3 loop and β-sheets, without displacing H12 from its agonist position. This result may explain kinetic differences between agonist and antagonist ligands observed for other nuclear receptors. The extended and flexible structure of RA initiated a methodological study in a simplified two-dimensional model system. The REMD force should in general be distributed to all atoms of the ligand to obtain the most unbiased results, but for a ligand which is tightly bound in the binding pocket through a strong electrostatic interaction, application of the REMD force on a single atom is preferred