Systematic identification of structure-specific protein-protein interactions.

The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis-mass spectrometry (LiP-MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP-MS detects well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson's disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.

Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase.

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement-a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound-and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.

Regulatory sites of CaM-sensitive adenylyl cyclase AC8 revealed by cryo-EM and structural proteomics.

Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gßγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

Structural insights into membrane adenylyl cyclases, initiators of cAMP signaling.

Membrane adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. As effector proteins of G protein-coupled receptors and other signaling pathways, ACs receive and amplify signals from the cell surface, translating them into biochemical reactions in the intracellular space and integrating different signaling pathways. Despite their importance in signal transduction and physiology, our knowledge about the structure, function, regulation, and molecular interactions of ACs remains relatively scarce. In this review, we summarize recent advances in our understanding of these membrane enzymes.

Structure of the connexin-43 gap junction channel in a putative closed state.

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.

Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels.

In myelinating Schwann cells, connection between myelin layers is mediated by gap junction channels (GJCs) formed by docked connexin 32 (Cx32) hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy without a cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high-resolution cryo-electron cryo-myography (cryo-EM) structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: In the W3S and R22G mutant HCs, the amino-terminal gating helix partially occludes the pore, consistent with a diminished HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.

Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel.

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.

Structural basis of organic cation transporter-3 inhibition.

Organic cation transporters (OCTs) facilitate the translocation of catecholamines, drugs and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 Å resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms.

Structure of Mycobacterium tuberculosis Cya, an evolutionary ancestor of the mammalian membrane adenylyl cyclases.

Mycobacterium tuberculosis adenylyl cyclase (AC) Rv1625c/Cya is an evolutionary ancestor of the mammalian membrane ACs and a model system for studies of their structure and function. Although the vital role of ACs in cellular signalling is well established, the function of their transmembrane (TM) regions remains unknown. Here, we describe the cryo-EM structure of Cya bound to a stabilizing nanobody at 3.6 Å resolution. The TM helices 1-5 form a structurally conserved domain that facilitates the assembly of the helical and catalytic domains. The TM region contains discrete pockets accessible from the extracellular and cytosolic side of the membrane. Neutralization of the negatively charged extracellular pocket Ex1 destabilizes the cytosolic helical domain and reduces the catalytic activity of the enzyme. The TM domain acts as a functional component of Cya, guiding the assembly of the catalytic domain and providing the means for direct regulation of catalytic activity in response to extracellular ligands.

Structural basis of adenylyl cyclase 9 activation.

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four conformations described here show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

Structural insights into an atypical secretory pathway kinase crucial for Toxoplasma gondii invasion.

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.

Cryo-EM structure of the Hedgehog release protein Dispatched.

The Hedgehog (Hh) signaling pathway controls embryonic development and adult tissue homeostasis in multicellular organisms. In Drosophila melanogaster, the pathway is primed by secretion of a dually lipid-modified morphogen, Hh, a process dependent on a membrane-integral protein Dispatched. Although Dispatched is a critical component of the pathway, the structural basis of its activity has, so far, not been described. Here, we describe a cryo-electron microscopy structure of the D. melanogaster Dispatched at 3.2-Å resolution. The ectodomains of Dispatched adopt an open conformation suggestive of a receptor-chaperone role. A three-dimensional reconstruction of Dispatched bound to Hh confirms the ability of Dispatched to bind Hh but using a unique mode distinct from those previously observed in structures of Hh complexes. The structure may represent the state of the complex that precedes shedding of Hh from the surface of the morphogen-releasing cell.

Affinity Purification of Membrane Proteins.

Biochemical, biophysical, and structural studies of membrane proteins rely on the availability of highly pure and monodisperse membrane protein samples. One of the most powerful methods for isolation of the membrane protein of interest is affinity purification. This methodology typically relies on engineering an affinity tag into the protein of interest and an affinity resin that specifically recognizes the tag, allowing one to purify the target protein in a single step. In some cases, the affinity purification procedure is combined with additional steps to increase the purity and homogeneity of the final protein sample. Here, we describe several protocols for affinity purification of TSPO, a small membrane protein. The techniques we use include immobilized metal affinity chromatography (IMAC) and strep-II tag-based streptavidin affinity chromatography.

Structural basis of sterol recognition by human hedgehog receptor PTCH1.

Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4-Å cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by 10 sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol-sensing domain (SSD) and the SSD-like domain (SSDL), which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.

Characterisation of the ligand binding sites in the translocator protein TSPO using the chimeric bacterial-mammalian constructs.

The translocator protein TSPO is in an important diagnostic and therapeutic target in a range of pathologies, including neuroinflammation and cancer. Despite the availability of several structures of TSPO homologues, our understanding of the molecular determinants that govern high-affinity interactions of TSPO with its ligands is incomplete. Here, in order to decipher the key structural elements of TSPO responsible for interactions with its ligands, we designed a panel of chimeric proteins mimicking the mammalian substrate binding site grafted onto the backbone of the Rhodobacter sphaeroides TSPO homologue, RsTSPO. One of the designed chimeric constructs, RsMouse, could be heterologously expressed and displayed improved binding affinities for the known TSPO drugs diazepam, PK11195 and NBD-FGIN-1-27. Furthermore, the chimeric protein had improved interactions with NBD-cholesterol, a fluorescent analogue of the presumed natural substrate of TSPO. Partial modifications of the transmembrane helix bundle in the chimeric construct differentially affected binding of the TSPO drugs and the natural substrates of TSPO, consistent with the presence of multiple ligand binding sites in the protein. Based on the available structures of TSPO homologues, the substrate interactions may involve a lateral opening of the protein in the TM1-3, and stabilisation of TM4-5 is important for drug-like ligand binding. These observations are consistent with our experimental results, which show that the determinants of high-affinity ligand interactions of TSPO are distinct for different classes of ligands.

The structure of a membrane adenylyl cyclase bound to an activated stimulatory G protein.

Membrane-integral adenylyl cyclases (ACs) are key enzymes in mammalian heterotrimeric GTP-binding protein (G protein)-dependent signal transduction, which is important in many cellular processes. Signals received by the G protein-coupled receptors are conveyed to ACs through G proteins to modulate the levels of cellular cyclic adenosine monophosphate (cAMP). Here, we describe the cryo-electron microscopy structure of the bovine membrane AC9 bound to an activated G protein αs subunit at 3.4-angstrom resolution. The structure reveals the organization of the membrane domain and helical domain that spans between the membrane and catalytic domains of AC9. The carboxyl-terminal extension of the catalytic domain occludes both the catalytic and the allosteric sites of AC9, inducing a conformation distinct from the substrate- and activator-bound state, suggesting a regulatory role in cAMP production.

Expression and purification of the mammalian translocator protein for structural studies.

The translocator protein (TSPO) is an 18 kDa polytopic membrane protein of the outer mitochondrial membrane, abundantly present in the steroid-synthesising cells. TSPO has been linked to a number of disorders, and it is recognised as a promising drug target with a range of potential medical applications. Structural and biochemical characterisation of a mammalian TSPO requires expression and purification of the protein of high quality in sufficiently large quantities. Here we describe detailed procedures for heterologous expression and purification of mammalian TSPO in HEK293 cells. We demonstrate that the established procedures can be used for untagged TSPO as well as for C-terminally fused TSPO constructs. Our protocol can be routinely used to generate high-quality TSPO preparations for biochemical and structural studies.

Role of the nucleotidyl cyclase helical domain in catalytically active dimer formation.

Nucleotidyl cyclases, including membrane-integral and soluble adenylyl and guanylyl cyclases, are central components in a wide range of signaling pathways. These proteins are architecturally diverse, yet many of them share a conserved feature, a helical region that precedes the catalytic cyclase domain. The role of this region in cyclase dimerization has been a subject of debate. Although mutations within this region in various cyclases have been linked to genetic diseases, the molecular details of their effects on the enzymes remain unknown. Here, we report an X-ray structure of the cytosolic portion of the membrane-integral adenylyl cyclase Cya from Mycobacterium intracellulare in a nucleotide-bound state. The helical domains of each Cya monomer form a tight hairpin, bringing the two catalytic domains into an active dimerized state. Mutations in the helical domain of Cya mimic the disease-related mutations in human proteins, recapitulating the profiles of the corresponding mutated enzymes, adenylyl cyclase-5 and retinal guanylyl cyclase-1. Our experiments with full-length Cya and its cytosolic domain link the mutations to protein stability, and the ability to induce an active dimeric conformation of the catalytic domains. Sequence conservation indicates that this domain is an integral part of cyclase machinery across protein families and species. Our study provides evidence for a role of the helical domain in establishing a catalytically competent dimeric cyclase conformation. Our results also suggest that the disease-associated mutations in the corresponding regions of human nucleotidyl cyclases disrupt the normal helical domain structure.