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Signaling pathways that mediate cell-cell communication are essential for collective cell behaviors in multicellular systems. The hedgehog (HH) pathway, first discovered and elucidated in Drosophila, is one of these iconic signaling systems that plays many roles during embryogenesis and in adults; abnormal HH signaling can lead to birth defects and cancer. We review recent structural and biochemical studies that have advanced our understanding of the vertebrate HH pathway, focusing on the mechanisms by which the HH signal is received by patched on target cells, transduced across the cell membrane by smoothened, and transmitted to the nucleus by GLI proteins to influence gene-expression programs.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Co-receptors add complexity to cell-cell signaling systems. The secreted semaphorin 3s (Sema3s) require a co-receptor, neuropilin (Nrp), to signal through plexin As (PlxnAs) in functions ranging from axon guidance to bone homeostasis, but the role of the co-receptor is obscure. Here we present the low-resolution crystal structure of a mouse semaphorin-plexin-Nrp complex alongside unliganded component structures. Dimeric semaphorin, two copies of plexin and two copies of Nrp are arranged as a dimer of heterotrimers. In each heterotrimer subcomplex, semaphorin contacts plexin, similar to in co-receptor-independent signaling complexes. The Nrp1s cross brace the assembly, bridging between sema domains of the Sema3A and PlxnA2 subunits from the two heterotrimers. Biophysical and cellular analyses confirm that this Nrp binding mode stabilizes a canonical, but weakened, Sema3-PlxnA interaction, adding co-receptor control over the mechanism by which receptor dimerization and/or oligomerization triggers signaling. © 2012 Nature America, Inc. All rights reserved.

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The Hedgehog signalling pathway is crucial for embryonic development and tissue homeostasis. Misregulation can result in severe developmental defects or oncogenesis, with an estimated 25% of all cancer deaths exhibiting improper Hedgehog signalling. The G-protein coupled receptor Smoothened plays a vital role in this pathway; it is responsible for transducing the Hedgehog signal across the plasma membrane. This makes Smoothened a highly attractive target for anticancer drug development. Despite the importance of Smoothened signalling and the plethora of structural data now available, the molecular mechanism of signal transduction is poorly understood. Here, we explore the mechanism of Smoothened activation and regulation by a range of small molecule ligands. We show that agonist binding at multiple sites can induce conformational changes required for effector binding. We couple these data with mutational analysis to propose distinct roles for each binding site. We also develop and validate a novel small molecule binding assay and adapt it to study allosteric communication within Smoothened. Finally, we present a strategy for use of the novel assay to identify next generation cancer treatments and address drug resistance.

Integral membrane proteins are responsible for a wide range of cellular functions and are important targets for the development of novel therapeutics. Therefore, obtaining structural and mechanistic insights into their function is of central importance for rational drug design. In this thesis, I focused on characterising three human integral membrane proteins using X-ray crystallography, cryo-EM, mass spectrometry and other biochemical methods. MFSD10 is a member of the drug:H+ antiporter family 1 (DHA1) of the Major Facilitator Superfamily of solute carriers and has been proposed to act as an efflux pump for organic anions, including several non-steroidal anti-inflammatory drugs (NSAIDs). Here, I present a crystal structure of human MFSD10 in an outward-facing partially occluded conformation, with a bound citrate ion from the crystallisation solution. The structure provides insights into the recognition of organic anions by MFSD10 and suggests a potential mechanism of proton coupling. An in vitro assay was employed to confirm the NSAID:H+ antiporter function of MFSD10 and will constitute the starting point for additional functional assays to dissect the mechanism of this transporter. The second focus of this thesis falls on elucidating the mechanisms of two enzyme families responsible for the acyl chain specificity in fatty acid elongation and sphingolipid biosynthesis, respectively. ELOVL elongases catalyse the first rate-limiting condensation step of the fatty acid elongation cycle in the endoplasmic reticulum (ER). The recently solved crystal structure of human ELOVL7 provided the first structural insights into this enzyme family. Here, I followed up on the structural data by characterising the mechanism of this enzyme using mass spectrometry. I demonstrated that catalysis by human ELOVL7, and likely other ELOVLs, proceeds via a ping-pong type mechanism involving an unusual covalent acyl-imidazole intermediate. Lastly, I focused on obtaining structural insights into the function of human ceramide synthases, which catalyse the N-acylation of sphingoid bases in the ER and determine the acyl chain composition of sphingolipids. I conducted a systematic screening of expression and purification conditions for two members of this family, identified suitable truncations for structural studies, obtained and validated nanobody binders, and optimised cryo-EM conditions. Together, these provide an advanced position for future structural studies to obtain valuable three-dimensional information on these important drug targets.

Members of the Hedgehog (HH) family of morphogenic signalling molecules are key mediators of many fundamental processes in embryonic development. A relatively small change in HH concentration results in the specification of distinct cell types. Such fine-tuning necessitates a range of regulatory cell-surface proteins to control the concentration of HH to which responding cells are exposed. This thesis focuses on the structural and functional characterisation of three human extracellular modulators of the HH pathway, namely the hedgehog-interacting protein HHIP, the glypican GPC3 and the Growth Arrest Specific 1 (GAS1). Cell culture robot protocols were developed to allow large-scale expression of these targets in mammalian cells (Chapter 3). In Chapter 4, the structure of the HH antagonist HHIP alone and in complex with HH ligands is described. These studies combined with functional experiments reveal a binding mode distinct from previously defined ligand interaction sites, with the HH metal-binding sites playing key roles. The structural and functional analysis of GPC3, another negative HH regulator, is described in Chapter 5. Binding studies suggest that the GPC3 core domain does not bind directly to SHH and that the GPC3-attched heparan sulphate chains play an important role in HH regulation. Chapter 6 reveals crystal structures of the GAS1 ectodomain, a HH agonist, allowing comparison to glial cell line-derived neurotrophic factor receptors, the identification of an unexpected ligand and mapping of the HH binding site. In summary, this work provides insights into the extracellular modulation of HH signalling and extends our current knowledge of this fundamental signalling pathway. It also offers a model to explain how both agonists and antagonists can adopt similar mechanisms of HH binding.

Neuronal synapses are the functional units of the nervous system, allowing the fast transmission of information between neurons. A plethora of proteins populate the cleft between connecting neurons, and many contribute to the formation of protein complexes than span the synaptic cleft. Such trans-synaptic complexes are of tremendous importance for synaptic function, affecting the neuronal wiring specificity, synaptic plasticity or the differentiation of the pre-and post-synaptic compartments. Two important protein families that form trans-synaptic complexes are ionotropic glutamate receptors (iGluRs) and receptor protein tyrosine phosphatases (RPTPs). In this thesis, I present work conducted on two prototypical protein complexes: the Glutamate Delta 2 (GluD2) receptor, in complex with Cerebellin 1 and b Neurexin 1; and the receptor protein tyrosine phosphatase μ (RPTPμ), which forms a homophilic dimer across cell contacts. Despite previous efforts, it is not well understood how these protein complexes are able to integrate their cell-adhesive properties with their downstream signalling functions. The aim of this thesis is to pave the way towards a mechanistic understanding of the function of these complexes. Using X-ray crystallography and biophysical assays, GluD2-specific nanobodies were identified, leading to the discovery of molecular tools that can stabilize distinct GluD2 conformations. Of particular interest, a nanobody capable of activating the receptor and preventing desensitization was identified, which will be instrumental for unveiling the GluD2 metabotropic signalling mechanism. Single-particle cryo-EM was applied to study the full- length GluD2 receptor, providing the first insights into the general architecture of Delta subtype iGluRs and laying the grounds to obtain a high resolution structure. RPTPμ- mediated cell-cell contacts were analysed by cryo electron tomography, obtaining data from cells thinned by focused ion beam (FIB) milling. The FIB milled sections revealed unprecedented detail of the architecture of insect cells and allowed the analysis of RPTPμ in a native environment. Subtomogram averaging shed light on a potential higher-order organization of RPTPμ at cell-cell contacts. Finally, a protocol for the purification and crystallization of the complete cytoplasmic region of RPTPμ was developed. This data paves the way towards unravelling the structure of the juxtamembrane domain, an essential part for understanding how type IIb RPTPs are regulated.

The Hedgehog signalling pathway is an essential developmental pathway present in all bilaterians that is involved in embryogenesis, body patterning and stem cell homeostasis. Dysregulation of the Hh pathway leads to various kinds of cancer, such as basal cell carcinoma and medulloblastoma. Smoothened (SMO), a Frizzled-type G-protein coupled receptor (GPCR), is the essential transmembrane signal transducer within the Hh pathway, conveying the signal from the upstream transmembrane protein, Patched1 (Ptc1), to the downstream intracellular proteins. The mechanisms by which SMO transmits the Hh signal from the extracellular environment, through the plasma membrane and to the intracellular proteins are not known. In this thesis, I present my work into the structural and functional characterisation of the extracellular and transmembrane domains (TMD) of human SMO in order to better understand the molecular mechanisms of its signal transduction. The extracellular region of SMO contains a highly conserved cysteine-rich domain (CRD) and a linker domain (LD). I present the first crystal structure of the CRD, LD and TMD of SMO, which is also the first crystal structure of a GPCR with a large functional extracellular domain. This structure revealed a domain architecture for SMO that enables regulation of its transmembrane domain by its extracellular domains. It also revealed a cholesterol molecule bound to the CRD, which we subsequently determined to be a new endogenous small-molecule agonist for SMO. I present five further structures of SMO bound to different small molecule agonists and antagonists. Together, these structures demonstrate that the position of the CRD relative to the TMD reflects the activation state of SMO. We also generated nanobodies against the extracellular region of SMO in order to stabilise its conformation. These studies not only improve our understanding of the workings of a key transmembrane protein within a fundamental signalling pathway but will also aid efforts to develop better therapeutics for an important cancer target.

The Hedgehog (Hh) pathway is a morphogen pathway essential for proper embryonic development of all bilaterians that remains active in adults where it contributes to tissue homoeostasis. Malfunction of the pathway results in developmental defect, while constitutive activation was correlated with multiple cancers. The Hh pathway is a validated cancer drug target with two FDA approved inhibitors targeting the Hh GPCR signal transducer Smoothened. This thesis focuses on two alternative Hh targets, the Sonic Hh (ShhN) morphogen and its acyltransferase Skinny Hh. In Chapter 2, the aim was to structurally characterise a complex between ShhN and its only small-molecule inhibitor Robotnikinin using in silico docking and crystallography. The results revealed that the most likely binding site of Robotnikinin is the pseudo-active site of ShhN and that the interactions involve the His134-Ser136 loop. In Chapter 3, the aim was to identify novel ShhN inhibitors using crystallographic fragment-based screening of 1,200 unique fragments. The screening resulted in 13 potential hits, 5 of which were found within the ion binding cleft. In Chapter 4, the aim was to validate the obtained hits and conduct preliminary structure activity relationship (SAR) assays. Based on the results, FRG 155 was identified as the preferred hit that bound to the the ShhN pseudo-active site with low micromolar affinity. Preliminary SAR identified positive and negative features of the fragment. In Chapter 5, the aim was to structurally and functionally characterise Skinny Hh. Protocols for successful expression and purification of milligram quantities of thermostable and active Skinny Hh were developed, providing a platform for crystallisation and drug screening. The first luminal loop of Skinny Hh was found to be essential for the activity of Skinny Hh. In summary, alternative Hh pathway targets, ShhN and Skinny Hh, were characterised using structural biology approaches and will contribute to the development of novel inhibitors with the application in Hh ligand-dependent tumours.

Cell-to-cell communication is vital to the morphogenesis and homeostasis of multicellular organisms and is linked to a variety of diseases. Extracellular Cysteine-Rich Domains (CRDs) are involved in the control of fundamental signalling pathways, mediating crucial interactions with protein and small molecule ligands. The localisation of extracellular ligands and receptors is regulated by the cell surface glycosaminoglycan (GAG) layer. In this thesis, structural and biochemical methods have been used to characterise two multi-domain proteins containing CRDs – HHIP and ROR2. In Chapter 3, the process of structure solution of the HHIP N-terminal CRD (HHIP-N) by Sulphur-SAD phasing techniques is described. HHIP-N is a novel CRD, with implications for small molecule binding present. Chapter 4 concerns the study of the regulation of HHIP secretion and its interaction with GAGs. Discrete GAG-binding motifs are found on the surfaces of HHIP-N and the C-terminal region of HHIP (HHIP-C). Structural studies with GAGs enabled snapshots of cell surface-bound states. Interactions were dissected using cellular assays and GAG-binding experiments. In addition, the dynamics of HHIP oligomerisation were analysed using solution scattering and ultracentrifugation techniques. Work in this chapter represents a complex mode of HHIP assembly regulated by GAGs. In chapter 5, structural and biophysical studies of ROR2 were performed. A structure of the ROR2 CRD with its C-terminal Kringle domain was determined. The structure forms a potentially stable structural unit with a marked inter-domain interface. The CRD structure is distinct from related CRDs and suggests that it cannot directly bind Wnt proteins. This was complemented by biophysical and cellular assays. In summary, the CRD is a fold found in two unrelated signalling pathways tied to a number of diseases and its role in multi-domain protein architectures has been characterised within this thesis. CRDs are involved in a wide range of interactions and these are essential for the precise regulation of signalling pathway activation.