Contactin 2 homophilic adhesion structure and conformational plasticity.

The cell-surface attached glycoprotein contactin 2 is ubiquitously expressed in the nervous system and mediates homotypic cell-cell interactions to organize cell guidance, differentiation, and adhesion. Contactin 2 consists of six Ig and four fibronectin type III domains (FnIII) of which the first four Ig domains form a horseshoe structure important for homodimerization and oligomerization. Here we report the crystal structure of the six-domain contactin 2Ig1-6 and show that the Ig5-Ig6 combination is oriented away from the horseshoe with flexion in interdomain connections. Two distinct dimer states, through Ig1-Ig2 and Ig3-Ig6 interactions, together allow formation of larger oligomers. Combined size exclusion chromatography with multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS) and native MS analysis indicates contactin 2Ig1-6 oligomerizes in a glycan dependent manner. SAXS and negative-stain electron microscopy reveals inherent plasticity of the contactin 2 full-ectodomain. The combination of intermolecular binding sites and ectodomain plasticity explains how contactin 2 can function as a homotypic adhesion molecule in diverse intercellular environments.

Structural insights into the contactin 1 - neurofascin 155 adhesion complex.

Cell-surface expressed contactin 1 and neurofascin 155 control wiring of the nervous system and interact across cells to form and maintain paranodal myelin-axon junctions. The molecular mechanism of contactin 1 - neurofascin 155 adhesion complex formation is unresolved. Crystallographic structures of complexed and individual contactin 1 and neurofascin 155 binding regions presented here, provide a rich picture of how competing and complementary interfaces, post-translational glycosylation, splice differences and structural plasticity enable formation of diverse adhesion sites. Structural, biophysical, and cell-clustering analysis reveal how conserved Ig1-2 interfaces form competing heterophilic contactin 1 - neurofascin 155 and homophilic neurofascin 155 complexes whereas contactin 1 forms low-affinity clusters through interfaces on Ig3-6. The structures explain how the heterophilic Ig1-Ig4 horseshoe's in the contactin 1 - neurofascin 155 complex define the 7.4 nm paranodal spacing and how the remaining six domains enable bridging of distinct intercellular distances.

Site-specific functionality and tryptophan mimicry of lipidation in tetraspanin CD9.

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell-cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation.

Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b.

Properdin enhances complement-mediated opsonization of targeted cells and particles for immune clearance. Properdin occurs as dimers, trimers and tetramers in human plasma, which recognize C3b-deposited surfaces, promote formation, and prolong the lifetime of C3bBb-enzyme complexes that convert C3 into C3b, thereby enhancing the complement-amplification loop. Here, we report crystal structures of monomerized properdin, which was produced by co-expression of separate N- and C-terminal constructs that yielded monomer-sized properdin complexes that stabilized C3bBb. Consistent with previous low-resolution X-ray and EM data, the crystal structures revealed ring-shaped arrangements that are formed by interactions between thrombospondin type-I repeat (TSR) domains 4 and 6 of one protomer interacting with the N-terminal domain (which adopts a short transforming-growth factor B binding protein-like fold) and domain TSR1 of a second protomer, respectively. Next, a structure of monomerized properdin in complex with the C-terminal domain of C3b showed that properdin-domain TSR5 binds along the C-terminal α-helix of C3b, while two loops, one from domain TSR5 and one from TSR6, extend and fold around the C3b C-terminus like stirrups. This suggests a mechanistic model in which these TSR5 and TSR6 "stirrups" bridge interactions between C3b and factor B or its fragment Bb, and thereby enhance formation of C3bB pro-convertases and stabilize C3bBb convertases. In addition, properdin TSR6 would sterically block binding of the protease factor I to C3b, thus limiting C3b proteolytic degradation. The presence of a valine instead of a third tryptophan in the canonical Trp-ladder of TSR domains in TSR4 allows a remarkable ca. 60°-domain bending motion of TSR4. Together with variable positioning of TSR2 and, putatively, TSR3, this explains the conformational flexibility required for properdin to form dimers, trimers, and tetramers. In conclusion, the results indicate that binding avidity of oligomeric properdin is needed to distinguish surface-deposited C3b molecules from soluble C3b or C3 and suggest that properdin-mediated interactions bridging C3b-B and C3b-Bb enhance affinity, thus promoting convertase formation and stabilization. These mechanisms explain the enhancement of complement-mediated opsonization of targeted cells and particle for immune clearance.

Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.

Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe3+ and Cu2+ ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe3+-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets.

Stochastic palmitoylation of accessible cysteines in membrane proteins revealed by native mass spectrometry.

Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. However, how specificity of palmitoylation and multiple palmitoylations in membrane proteins are determined is not well understood. Here, we profile palmitoylation states of three human claudins, human CD20 and cysteine-engineered prokaryotic KcsA and bacteriorhodopsin by native mass spectrometry. Cysteine scanning of claudin-3, KcsA, and bacteriorhodopsin shows that palmitoylation is independent of a sequence motif. Palmitoylations are observed for cysteines exposed on the protein surface and situated up to 8 Å into the inner leaflet of the membrane. Palmitoylation on multiple sites in claudin-3 and CD20 occurs stochastically, giving rise to a distribution of palmitoylated membrane-protein isoforms. Non-native sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites. These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif.

Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses.

The complement system labels microbes and host debris for clearance. Degradation of surface-bound C3b is pivotal to direct immune responses and protect host cells. How the serine protease factor I (FI), assisted by regulators, cleaves either two or three distant peptide bonds in the CUB domain of C3b remains unclear. We present a crystal structure of C3b in complex with FI and regulator factor H (FH; domains 1-4 with 19-20). FI binds C3b-FH between FH domains 2 and 3 and a reoriented C3b C-terminal domain and docks onto the first scissile bond, while stabilizing its catalytic domain for proteolytic activity. One cleavage in C3b does not affect its overall structure, whereas two cleavages unfold CUB and dislodge the thioester-containing domain (TED), affecting binding of regulators and thereby determining the number of cleavages. These data explain how FI generates late-stage opsonins iC3b or C3dg in a context-dependent manner, to react to foreign, danger or healthy self signals.

Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode.

Regulators of complement activation (RCA) inhibit complement-induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i-iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b-binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease-related mutations and immune evasion.

Structures of Wnt-antagonist ZNRF3 and its complex with R-spondin 1 and implications for signaling.

Zinc RING finger 3 (ZNRF3) and its homolog RING finger 43 (RNF43) antagonize Wnt signaling in adult stem cells by ubiquitinating Frizzled receptors (FZD), which leads to endocytosis of the Wnt receptor. Conversely, binding of ZNRF3/RNF43 to LGR4-6 - R-spondin blocks Frizzled ubiquitination and enhances Wnt signaling. Here, we present crystal structures of the ZNRF3 ectodomain and its complex with R-spondin 1 (RSPO1). ZNRF3 binds RSPO1 and LGR5-RSPO1 with micromolar affinity via RSPO1 furin-like 1 (Fu1) domain. Anonychia-related mutations in RSPO4 support the importance of the observed interface. The ZNRF3-RSPO1 structure resembles that of LGR5-RSPO1-RNF43, though Fu2 of RSPO1 is variably oriented. The ZNRF3-binding site overlaps with trans-interactions observed in 2:2 LGR5-RSPO1 complexes, thus binding of ZNRF3/RNF43 would disrupt such an arrangement. Sequence conservation suggests a single ligand-binding site on ZNRF3, consistent with the proposed competing binding role of ZNRF3/RNF43 in Wnt signaling.

Structure of stem cell growth factor R-spondin 1 in complex with the ectodomain of its receptor LGR5.

Leucine-rich repeat-containing G protein-coupled receptors 4-6 (LGR4-LGR6) are receptors for R-spondins, potent Wnt agonists that exert profound trophic effects on Wnt-driven stem cells compartments. We present crystal structures of a signaling-competent fragment of R-spondin 1 (Rspo1) at a resolution of 2.0 Å and its complex with the LGR5 ectodomain at a resolution of 3.2 Å. Ecto-LGR5 binds Rspo1 at its concave leucine-rich-repeat (LRR) surface, forming a dimeric 2:2 complex. Fully conserved residues on LGR4-LGR6 explain promiscuous binding of R-spondins. A phenylalanine clamp formed by Rspo1 Phe106 and Phe110 pinches Ala190 of LGR5 and is critical for binding. Mutations related to congenital anonychia reduce signaling, but not binding of Rspo1 to LGR5. Furthermore, antibody binding to the extended loop of the C-terminal LRR cap of LGR5 activates signaling in a ligand-independent manner. Thus, our data reveal binding of R-spondins to conserved sites on LGR4-LGR6 and, in analogy to FSHR and related receptors, suggest a direct signaling role for LGR4-LGR6 in addition to its formation of Wnt receptor and coreceptor complexes.

Functional differences of invariant and highly conserved residues in the extracellular domain of the glycoprotein hormone receptors.

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in ß-strands 2 (Lys(74)), 4 (Tyr(124), Asn(129), and Thr(130)), and 5 (Asp(150) and Asp(153)) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in ß-strands 2 (Ser(78)) and 8 (Asp(224) and Ser(226)) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.

Leydig cells express follicle-stimulating hormone receptors in African catfish.

This report aimed to establish, using African catfish, Clarias gariepinus, as model species, a basis for understanding a well-known, although not yet clarified, feature of male fish reproductive physiology: the strong steroidogenic activity of FSHs. Assays with gonadotropin receptor-expressing cell lines showed that FSH activated its cognate receptor (FSHR) with an at least 1000-fold lower EC50 than when challenging the LH receptor (LHR), whereas LH stimulated both receptors with similar EC50s. In androgen release bioassays, FSH elicited a significant response at lower concentrations than those required to cross-activate of the LHR, indicating that FSH stimulated steroid release via FSHR-dependent mechanisms. LHR/FSHR-mediated stimulation of androgen release was completely abolished by H-89, a specific protein kinase A inhibitor, pointing to the cAMP/protein kinase A pathway as the main route for both LH- and FSH-stimulated steroid release. Localization studies showed that intratubular Sertoli cells express FSHR mRNA, whereas, as reported for the first time in a vertebrate, catfish Leydig cells express both LHR and FSHR mRNA. Testicular FSHR and LHR mRNA expression increased gradually during pubertal development. FSHR, but not LHR, transcript levels continued to rise between completion of the first wave of spermatogenesis at about 7 months and full maturity at about 12 months of age, which was associated with a previously recorded approximately 3-fold increase in the steroid production capacity per unit testis weight. Taken together, our data strongly suggest that the steroidogenic potency of FSH can be explained by its direct trophic action on FSHR-expressing Leydig cells.

Identification of a luteinizing hormone-selective determinant in the exodomain of a follicle-stimulating hormone receptor.

Mammalian glycoprotein hormone receptors (GpHRs) display a stringent selectivity for their cognate hormones. In contrast, the follicle-stimulating hormone receptor of the African catfish (cfFSHR) is promiscuously activated by catfish luteinizing hormone (cfLH). Glycoprotein hormones bind to the concave site of the cusp-shaped N-terminal GpHR exodomain, which is formed by 9-10 parallel beta-strands. Hence, hormone selectivity of each GpHR for its cognate ligand is defined by amino acid sequence divergence in these beta-strands between different GpHRs. To identify the molecular determinants that allow promiscuous activation of the cfFSHR by cfLH, beta-strands were systematically exchanged between the cfFSHR and the human FSHR. Both gain-of-function and loss-of-function mutational approaches revealed that beta-strand 2 of the cfFSHR contains determinants that contribute to the receptor's responsiveness to cfLH.

Identification of follicle-stimulating hormone-selective beta-strands in the N-terminal hormone-binding exodomain of human gonadotropin receptors.

Glycoprotein hormone receptors contain large N-terminal extracellular domains (ECDs) that distinguish these receptors from most other G protein-coupled receptors. Each glycoprotein hormone receptor ECD consists of a curved leucine-rich repeat domain flanked by N- and C-terminal cysteine-rich regions. Selectivity of the different glycoprotein hormone receptors for their cognate hormones is exclusively determined by their ECDs and, in particular, their leucine-rich repeat domain. To identify human (h)FSH-selective determinants we used a gain-of-function mutagenesis strategy in which beta-strands of the hLH receptor (hLH-R) were substituted with their hFSH receptor (hFSH-R) counterparts. Introduction of hFSH-R beta-strand 1 into hLH-R conferred responsiveness to hFSH, whereas hLH-R mutants harboring one of the other hFSH-R beta-strands displayed none or very limited sensitivity to hFSH. However, combined substitution of hFSH-R beta-strand 1 and some of the other hFSH-R beta-strands further increased the sensitivity of the mutant hLH-R to hFSH. The apparent contribution of multiple hFSH-R beta-strands in providing a selective hormone binding interface corresponds well with their position in relation to hFSH as recently determined in the crystal structure of hFSH in complex with part of the hFSH-R ECD.

Fish FSH receptors bind LH: how to make the human FSH receptor to be more fishy?

In mammals, the interactions between glycoprotein hormones and their cognate receptors are highly specific; unintended cross-reactivity under normal physiological conditions has not been observed. The interactions between fish gonadotropins and their receptors, on the other hand, appeared to be less discriminatory. For example, the catfish follicle-stimulating hormone (FSH) receptor was highly responsive to both catfish luteinizing hormone (LH) and catfish FSH. Similarly, the FSH receptor of coho salmon bound both salmon FSH and LH. In contrast, LH receptors of both species were found to be rather specific for their cognate LH. This paper intends to summarize the current situation with special emphasis to our comparative structure-function studies that aim at elucidating the molecular basis of ligand selectivity (in mammals) and ligand promiscuity (in fish).

Receptor-selective determinants in catfish gonadotropin seat-belt loops.

Mammalian gonadotropins are highly selective. Charge differences between the Cys(10-11) sequence of FSHbeta and LHbeta/CGbeta seat-belt loops determine the ability of these hormones to interact with the LH-R. Selective FSH-R binding is mainly dependent on the presence of an FSHbeta-specific sequence between Cys(11-12) of the seat-belt loop. Intriguingly, African catfish LHbeta (cfLHbeta) lacks a positively charged Cys(10-11) region and stimulates both catfish LH-R and FSH-R with comparable potencies. Our studies on the promiscuous behaviour of cfLH using chimeric gonadotropins revealed that the Cys(10-11) region of cfLHbeta contains cfLH-R-selective determinants, whereas the Cys(11-12) region of cfLHbeta confers FSH-R-stimulating activity to cfLH. Hence, the location of receptor-selective determinants appeared to be fairly well conserved throughout evolution, despite the low sequence identity between mammalian and catfish seat-belt loops. Moreover, various structure-function differences between gonadotropins are discussed in the context of the different (female) reproductive strategies between mammalian and non-mammalian species that required the divergence to a more specific LH-R-stimulating activity of one of the gonadotropins in mammals.

Opposite contribution of two ligand-selective determinants in the N-terminal hormone-binding exodomain of human gonadotropin receptors.

The nine leucine-rich repeat-containing exodomains of the human FSH receptor (hFSH-R) and the human LH/chorionic gonadotropin receptor (hLH-R) harbor molecular determinants that allow the mutually exclusive binding of human FSH (hFSH) and human LH (hLH)/human chorionic gonadotropin (hCG) when these hormones are present in physiological concentrations. Previously, we have shown that the beta-strands of hLH-R leucine-rich repeats 3 and 6 can confer full hCG/hLH responsiveness and binding when simultaneously introduced into a hFSH-R background without affecting the receptor's responsiveness to hFSH. In the present study, we have determined the nature of contribution of each of these two beta-strands in conferring hCG/hLH responsiveness to this mutant hFSH-R. Human LH-R beta-strand 3 appeared to function as a positive hCG/hLH determinant by increasing the hCG/hLH responsiveness of the hFSH-R. In contrast, mutagenesis of hFSH-R beta-strand 6, rather than the introduction of its corresponding hLH-R beta-strand, appeared to allow the interaction of hCG/hLH with the hFSH-R. Hence, hFSH-R beta-strand 6 functions as a negative determinant and, as such, restrains binding of hCG/hLH to the hFSH-R. Detailed mutagenic analysis revealed that the ability of the hFSH-R to interact with hCG/hLH depends primarily on the identity of two amino acids (Asn104, a positive LH-R determinant, and Lys179 a negative FSH-R determinant) that are situated on the C-terminal ends of beta-strands 3 and 6, respectively.

Ligand selectivity of gonadotropin receptors. Role of the beta-strands of extracellular leucine-rich repeats 3 and 6 of the human luteinizing hormone receptor.

The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their approximately 350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeat-containing subdomain (LRR subdomain) flanked by N- and C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel beta-strands. Receptors in which individual beta-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R beta-strands 3 and 6. A mutant receptor in which the hFSH-R beta-strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most beta-strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.