FSH, a glycoprotein hormone, and the FSH receptor (FSHR), a G protein-coupled receptor, play central roles in human reproduction. We report the crystal structure of FSH in complex with the entire extracellular domain of FSHR (FSHR ED ), including the enigmatic hinge region that is responsible for signal specificity. Surprisingly, the hinge region does not form a separate structural unit as widely anticipated but is part of the integral structure of FSHR ED . In addition to the known hormone-binding site, FSHR ED provides interaction sites with the hormone: a sulfotyrosine (sTyr) site in the hinge region consistent with previous studies and a potential exosite resulting from putative receptor trimerization. Our structure, in comparison to others, suggests FSHR interacts with its ligand in two steps: ligand recruitment followed by sTyr recognition. FSH first binds to the high-affinity hormone-binding subdomain of FSHR and reshapes the ligand conformation to form a sTyr-binding pocket. FSHR then inserts its sTyr (i.e., sulfated Tyr335) into the FSH nascent pocket, eventually leading to receptor activation.F SH is a gonadotropin that stimulates steroidogenesis and gametogenesis in the gonads. Secreted by the anterior pituitary gland, FSH regulates the menstrual cycle and ovarian follicular maturation in women and supports sperm production in men. FSH acts by binding to the FSH receptor (FSHR) on the granulosa cell surface in ovaries and the Sertoli cell surface in testes. The stimulated receptor leads to the dissociation of α-and βγ-subunits of G protein heterotrimer inside the cell. The α-subunit activates adenylyl cyclase, resulting in an increase of cAMP levels, and ultimately leads to the increased steroid production that is necessary for follicular growth and ovulation in women. The free βγ dimers recruit G protein-coupled receptor (GPCR) kinases to the receptor, which, in turn, lead to the recruitment of β-arrestin to the receptor (1). FSH is used clinically for controlled ovarian stimulation in women treated with assisted reproductive technologies and also for the treatment of anovulatory infertility in women and hypogonadotropic hypogonadism in men. The central role of FSH in human reproduction makes its receptor a unique pharmaceutical target in the field of fertility regulation (2-4).The glycoprotein hormone (GPH) family has four members: FSH; two other pituitary hormones, luteinizing hormone and thyroid-stimulating hormone (TSH); and one placental hormone, chorionic gonadotropin. The four members are homologous in sequence, structure, and function. Each member is a heterodimer composed of a common α-subunit and a hormone-specific β-subunit. The crystal structures of FSH and human CG (hCG) revealed that both α-and β-subunits adopt similar folds of cystineknot architecture (5-7). The assembled α-and β-heterodimers bind to their respective receptors with high affinity and hormone specificity, resulting in similar signaling pathways but distinct biological responses.

Inhibitor of κB (IκB) kinase (IKK) phosphorylates IκB proteins leading to their degradation and liberation of nuclear factor κB (NF-κB) for gene transcription. Here we report the crystal structure of IKKβ in complex with an inhibitor at 3.6 Å resolution. The structure reveals a tri-modular architecture with the kinase domain (KD), a ubiquitin-like domain (ULD) and an elongated, α-helical scaffold/dimerization domain (SDD). Surprisingly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of SDD. The ULD and SDD mediate a critical interaction with IκBα that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKKβ dimerization, but dimerization per se is not important for maintaining IKKβ activity, and instead is required for IKKβ activation. Other IKK family members IKKα, TBK1 and IKKi may share the similar tri-modular architecture and function.

This article reviews the progress made in the field of glycoprotein hormones (GPH) and their receptors (GPHR) by several groups of structural biologists including ourselves aiming to gain insight into GPH signaling mechanisms. The GPH family consists of four members, with follicle-stimulating hormone (FSH) being the prototypic member. GPH members belong to the cystine-knot growth factor superfamily, and their receptors (GPHR), possessing unusually large N-terminal ectodomains, belong to the G-protein coupled receptor Family A. GPHR ectodomains can be divided into two subdomains: a high-affinity hormone binding subdomain primarily centered on the N-terminus, and a second subdomain that is located on the C-terminal region of the ectodomain that is involved in signal specificity. The two subdomains unexpectedly form an integral structure comprised of leucine-rich repeats (LRRs). Following the structure determination of hCG in 1994, the field of FSH structural biology has progressively advanced. Initially, the FSH structure was determined in partially glycosylated free form in 2001, followed by a structure of FSH bound to a truncated FSHR ectodomain in 2005, and the structure of FSH bound to the entire ectodomain in 2012. Comparisons of the structures in three forms led a proposal of a two-step monomeric receptor activation mechanism. First, binding of FSH to the FSHR high-affinity hormone-binding subdomain induces a conformational change in the hormone to form a binding pocket that is specific for a sulfated-tyrosine found as sTyr 335 in FSHR. Subsequently, the sTyr is drawn into the newly formed binding pocket, producing a lever effect on a helical pivot whereby the docking sTyr provides as the 'pull & lift' force. The pivot helix is flanked by rigid LRRs and locked by two disulfide bonds on both sides: the hormone-binding subdomain on one side and the last short loop before the first transmembrane helix on the other side. The lift of the sTyr loop frees the tethered extracellular loops of the 7TM domain, thereby releasing a putative inhibitory influence of the ectodomain, ultimately leading to the activating conformation of the 7TM domain. Moreover, the data lead us to propose that FSHR exists as a trimer and to present an FSHR activation mechanism consistent with the observed trimeric crystal form. A trimeric receptor provides resolution of the enigmatic, but important, biological roles played by GPH residues that are removed from the primary FSH-binding site, as well as several important GPCR phenomena, including negative cooperativity and asymmetric activation. Further reflection pursuant to this review process revealed additional novel structural characteristics such as the identification of a 'seat' sequence in GPH. Together with the 'seatbelt', the 'seat' enables a common heteodimeric mode of association of the common α subunit non-covalently and non-specifically with each of the three different β subunits. Moreover, it was possible to establish a dimensional order that can be used to estimate LRR curvatures...

Amino acid sequence alignments of cholinesterases revealed that 6 of 14 aromatic amino acid residues lining the active center gorge of acetylcholinesterase are replaced by aliphatic amino acid residues in butyrylcholinesterase. The Y337 (F330) in mammalian acetylcholinesterase, which is replaced by A328 in human butyrylcholinesterase, is implicated in the binding of ligands such as huperzine A, edrophonium, and acridines and one end of bisquaternary compounds such as BW284C51 and decamethonium. Y337 may sterically hinder the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution. Inhibition studies of (-)-huperzine A with human butyrylcholinesterase mutants, where A328 (KI = 194.6 microM) was modified to either F (KI = 0.6 microM, as in Torpedo acetylcholinesterase) or Y (KI = 0.032 microM, as in mammalian acetylcholinesterase), confirmed previous observations made with acetylcholinesterase mutants that this residue is important for binding huperzine A. Inhibition studies of ethopropazine with butyrylcholinesterase mutants, where A328 (KI = 0.18 microM) was modified to either F (KI = 0.82 microM) or Y (KI = 0.28 microM), suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active site gorge showed that the poor inhibitory activity of ethopropazine toward acetylcholinesterase was due to the smaller dimension of the active site gorge which was unable to accommodate the bulky inhibitor molecule. The volume of the butyrylcholinesterase active site gorge is approximately 200 A3 larger than that of the acetylcholinesterase gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations.