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Structural basis of semaphorin‐plexin cis interaction

2020; Springer Nature; Volume: 39; Issue: 13 Linguagem: Inglês

10.15252/embj.2019102926

1460-2075

Daniel Rozbeský, Marieke G. Verhagen, Dimple Karia, Gergely Nagy, Luis Álvarez, R.A. Robinson, Karl Harlos, Sergi Padilla‐Parra, R. Jeroen Pasterkamp, E. Yvonne Jones,

Zebrafish Biomedical Research Applications

Article5 June 2020Open Access Transparent process Structural basis of semaphorin-plexin cis interaction Daniel Rozbesky Daniel Rozbesky orcid.org/0000-0001-6546-8219 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Marieke G Verhagen Marieke G Verhagen Department of Translational Neuroscience, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Dimple Karia Dimple Karia Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Gergely N Nagy Gergely N Nagy Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Luis Alvarez Luis Alvarez Cellular Imaging, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Ross A Robinson Ross A Robinson Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Karl Harlos Karl Harlos orcid.org/0000-0002-7266-4354 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Sergi Padilla-Parra Sergi Padilla-Parra Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Cellular Imaging, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author R Jeroen Pasterkamp Corresponding Author R Jeroen Pasterkamp [email protected] orcid.org/0000-0003-1631-6440 Department of Translational Neuroscience, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Edith Yvonne Jones Corresponding Author Edith Yvonne Jones [email protected] orcid.org/0000-0002-3834-1893 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Daniel Rozbesky Daniel Rozbesky orcid.org/0000-0001-6546-8219 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Marieke G Verhagen Marieke G Verhagen Department of Translational Neuroscience, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Dimple Karia Dimple Karia Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Gergely N Nagy Gergely N Nagy Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Luis Alvarez Luis Alvarez Cellular Imaging, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Ross A Robinson Ross A Robinson Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Karl Harlos Karl Harlos orcid.org/0000-0002-7266-4354 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Sergi Padilla-Parra Sergi Padilla-Parra Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Cellular Imaging, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author R Jeroen Pasterkamp Corresponding Author R Jeroen Pasterkamp [email protected] orcid.org/0000-0003-1631-6440 Department of Translational Neuroscience, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Edith Yvonne Jones Corresponding Author Edith Yvonne Jones [email protected] orcid.org/0000-0002-3834-1893 Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK Search for more papers by this author Author Information Daniel Rozbesky1, Marieke G Verhagen2, Dimple Karia1, Gergely N Nagy1, Luis Alvarez3, Ross A Robinson1,4, Karl Harlos1, Sergi Padilla-Parra1,3,5,6, R Jeroen Pasterkamp *,2 and Edith Yvonne Jones *,1 1Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK 2Department of Translational Neuroscience, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands 3Cellular Imaging, Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK 4Present address: Immunocore Ltd, Abingdon, UK 5Present address: Department of Infectious Diseases, Faculty of Life Sciences & Medicine, King's College London, London, UK 6Present address: Randall Centre for Cell and Molecular Biophysics, King's College London, London, UK *Corresponding author. Tel: +31 88756 8831; E-mail: [email protected] author. Tel: +44 1865 287547; E-mail: [email protected] The EMBO Journal (2020)39:e102926https://doi.org/10.15252/embj.2019102926 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Semaphorin ligands interact with plexin receptors to contribute to functions in the development of myriad tissues including neurite guidance and synaptic organisation within the nervous system. Cell-attached semaphorins interact in trans with plexins on opposing cells, but also in cis on the same cell. The interplay between trans and cis interactions is crucial for the regulated development of complex neural circuitry, but the underlying molecular mechanisms are uncharacterised. We have discovered a distinct mode of interaction through which the Drosophila semaphorin Sema1b and mouse Sema6A mediate binding in cis to their cognate plexin receptors. Our high-resolution structural, biophysical and in vitro analyses demonstrate that monomeric semaphorins can mediate a distinctive plexin binding mode. These findings suggest the interplay between monomeric vs dimeric states has a hereto unappreciated role in semaphorin biology, providing a mechanism by which Sema6s may balance cis and trans functionalities. Synopsis This study reveals that semaphorin-plexin binding in cis can be mediated by two independent binding modes that serve as an inhibitory mechanism to signalling in trans. These findings suggest the interplay between monomeric and dimeric semaphorins plays a central role in semaphorin biology. Drosophila Sema1b is a monomer on the cell surface and can bind in cis with PlexA. Sema1b can interact with PlexA using two independent modes, head-to-head or side-on. Sema1b-PlexA binding in cis inhibits PlexA signalling by dimeric Sema1a binding in trans. Mouse Sema6A utilises the same molecular mechanism for cis interaction with its cognate plexin receptor as its Drosophila homolog, Sema1b. Introduction Semaphorins and plexins are one of the classical cell guidance ligand-receptor families first characterised by their ability to steer axon growth cones in the developing nervous system (Kolodkin et al, 1992, 1993; Luo et al, 1993; Tamagnone et al, 1999). Beyond axon guidance, semaphorin–plexin signalling is implicated in a plethora of physiological functions including other aspects of neural development, angiogenesis, vascularisation, organogenesis and regulation of immune responses (Tran et al, 2007; Pasterkamp, 2012; Takamatsu & Kumanogoh, 2012). Conversely, deregulation of semaphorin–plexin signalling is associated with tumour progression and other diseases (Tamagnone, 2012). Exquisite control of the local level and biological consequence of signalling is characteristic of the semaphorin–plexin system and essential for many of its functions. Semaphorins are secreted, transmembrane or GPI-anchored proteins (Kolodkin et al, 1993). Membrane-attached semaphorins and plexins commonly function through cell-to-cell trans interactions in which the semaphorin ligands and plexin receptors are presented on opposing cells. However, when ligand and receptor are present on the same cell surface there is potential for ligand-receptor binding in cis at the same plasma membrane. An increasing body of evidence points to the importance of cis interactions in the regulation of diverse cell guidance signalling systems (Seiradake et al, 2016). In the semaphorin–plexin signalling system, cis interactions were first described between class 6 semaphorins (Sema6s) and their cognate plexin class A (PlxnA) receptors. Studies in migrating granule cells suggest that binding of Sema6A and PlxnA2 in cis inhibits the binding of PlxnA2 by Sema6A in trans as the absence of Sema6A in cis causes over-activation of PlxnA2 (Renaud et al, 2008). The cis interaction of Sema6A-PlxnA2 has been further reported to be essential for proper development of lamina-restricted projection of hippocampal mossy fibres (Suto et al, 2007; Tawarayama et al, 2010). Finally, the inhibitory effect of cis interaction has been demonstrated between Sema6A and PlxnA4 (Haklai-Topper et al, 2010), and Sema6B and PlxnA2 (Andermatt et al, 2014). Contrary to these inhibition effects, the cis interaction between semaphorin SMP-1 and the PlxnA4 homolog, PLX-1, in C. elegans has been shown to result in plexin activation (Mizumoto & Shen, 2013). Similarly, mouse Sema5A signals through PlxnA2 co-expressed on hippocampal dentate granule cells to regulate synaptogenesis (Duan et al, 2014). Perhaps the most exquisite interplay of semaphorin–plexin cis and trans interactions reported to date is that of Sema6A and PlxnA2 in the elaboration of dendritic arbors during retinal circuit assembly (Sun et al, 2013). Intriguingly, it has been suggested that the cis and trans interaction modes of semaphorins and plexins require distinct binding sites (Haklai-Topper et al, 2010; Perez-Branguli et al, 2016). The first crystal structures of semaphorins revealed that the hallmark N-terminal sema domain is a seven-bladed β-propeller with a propensity to dimerise (Antipenko et al, 2003; Love et al, 2003) and the homodimeric architecture has long been reported as essential for semaphorins to function as repulsive guidance cues (Klostermann et al, 1998; Koppel & Raper, 1998). In a recent study, we have demonstrated that semaphorins can also form heterodimers and monomers, and thus, their architecture is not restricted to homodimers (Rozbesky et al, 2019). Plexins are type I transmembrane proteins containing an N-terminal sema domain followed by multiple PSI and IPT domains in their extracellular segment (Bork et al, 1999; Tamagnone et al, 1999). The plexin intracellular region has a distinctive GAP domain architecture (He et al, 2009; Tong et al, 2009; Bell et al, 2011; Wang et al, 2013), which structural and functional studies suggest is activated by dimerisation (He et al, 2009; Tong et al, 2009; Wang et al, 2012, 2013). Recent crystal structures of full-length mouse PlxnA ectodomains (comprising ten domains) revealed a ring-like overall shape, which is presumably orientated parallel to the plane of the plasma membrane at the cell surface (Kong et al, 2016). The ring-like structure is consistent with an observed PlxnA-to-PlxnA "head-to-stalk" cis interaction being able to maintain pre-ligand bound plexins in a clustered, but autoinhibited, state on the cell surface, presumably by favouring separation, and thus preventing spontaneous dimerisation, of the transmembrane and intracellular regions (Kong et al, 2016). The existence of inactive dimers of pre-ligand bound plexin is further supported by data from fluorescence cross-correlation spectroscopy experiments on mouse PlxnA4 (Marita et al, 2015). Crystal structures have been reported for complexes formed between semaphorin ectodomains and fragments comprising up to four of the N-terminal domains of the cognate plexin ectodomain. These semaphorin–plexin complexes all show a bivalent 2:2 architecture that comprises a semaphorin dimer interacting with two copies of the plexin consistent with receptor activation by ligand-mediated dimerisation, a conclusion supported by structure-guided biophysical and cell-based assays (Janssen et al, 2010; Liu et al, 2010; Nogi et al, 2010). In all semaphorin–plexin complexes analysed to date, the semaphorins and plexins bind in a head-to-head (semaphorin sema domain-to-plexin sema domain) orientation suitable for a trans interaction between ligands and receptors attached to opposing cell surfaces triggering receptor activation (Kong et al, 2016). No molecular interaction surfaces have been characterised in terms of their ability to mediate semaphorin–plexin binding modes in cis; thus, the structural basis and molecular mechanism(s) governing the divergent outcomes of cis and trans binding remain elusive. The ectodomain of Sema6A forms a weak dimer with monomeric and dimeric forms present in solution (Janssen et al, 2010; Nogi et al, 2010). The interplay of monomeric and dimeric Sema6 at the plasma membrane is likely relevant to cis interactions with the cognate PlxnA receptors. Structural and biophysical analyses at high concentrations have provided detailed insight into the interaction of dimeric Sema6A with PlxnA2; however, because of the monomer-dimer equilibrium, the binding properties of wild-type monomeric Sema6A have eluded direct analysis. In structural and biophysical studies of the Drosophila semaphorin system, we recently discovered a wild-type monomeric semaphorin, Sema1b (Rozbesky et al, 2019). This unexpected discovery provided us with a system in which we could dissect the interaction surfaces, and contributions to plexin binding in cis, of a semaphorin that is purely in the monomeric state. The class 1 (Sema1a and Sema1b) and class 2 (Sema2a and Sema2b) Drosophila semaphorins are membrane-attached and secreted, respectively. Sema1a and Sema1b are most closely related to the mammalian class 6 semaphorins and interact with the sole Drosophila class A plexin, PlexA (Pasterkamp, 2012). In previous studies, we have shown that the secreted Drosophila semaphorins, Sema2a and Sema2b, and also the ectodomain of membrane-attached Sema1aecto are disulphide-linked dimers. All three of these semaphorins contain an intermolecular sema-to-sema disulphide bridge. Conversely, we found the ectodomain of membrane-attached Sema1becto to be a monomer in solution due to an amino acid substitution in the intermolecular disulphide bridge at position 254 (Rozbesky et al, 2019). Here, we show that Drosophila Sema1b is a monomer on the cell surface and can interact in cis with PlexA. We further report two crystal structures of Sema1b complexed with the semaphorin-binding region of PlexA. The crystal structures, along with biophysical and cell-based assays, show that monomeric Sema1b binds PlexA at two independent binding sites. One interaction mode corresponds to the canonical head-to-head orientation described previously for semaphorin–plexin binding. The second mode uses an interactive surface on Sema1b that is occluded in dimeric semaphorins. We were able to demonstrate that this novel "side-on" binding mode perturbs the ring-like structure of the PlexA ectodomain. In cell collapse assays, we found that the side-on mode of monomeric Sema1b-PlexA binding in cis was sufficient to inhibit PlexA signalling by dimeric Sema1a binding in trans. In dorsal root ganglion neurons, we also confirmed that mouse Sema6A utilises the same molecular mechanism for cis interaction with its cognate plexin receptor as its Drosophila homolog, Sema1b. Based on our findings, we propose models for semaphorin–plexin cis interactions which incorporate a distinctive role for monomeric semaphorin binding in the regulation of plexin signalling. Results Sema1b is a monomer on the cell surface and fails to mediate PlexA dimerisation We considered the oligomeric state of Sema1b on the membrane of live cells. COS-7 cells were transiently transfected with Sema1b-F254C-mClover (a mutant which provides Sema1a-like disulphide-linked dimer formation) or with the wild-type Sema1b-mClover. Both constructs encompassed the ectodomain followed by a native transmembrane segment, short cytoplasmic linker and the C-terminal fluorescent protein mClover. mClover is a monomeric bright yellow-green fluorescent protein commonly used for the analysis of dimerisation or protein–protein interactions in live cells (Lam et al, 2012). Using Number and Brightness analysis, we determined a molecular brightness (ε) in live cells, which is directly related to the oligomeric state. Number and Brightness analysis is a fluorescence fluctuation spectroscopy technique to measure the average number and oligomeric state of labelled entities in each pixel of a stack of fluorescently labelled images (Digman et al, 2008). We have recently developed the method further by implementing a novel detrending algorithm to detect monomers and dimers in live cells (Nolan et al, 2017, 2018a; Iliopoulou et al, 2018) or in vitro (Nolan et al, 2018b). Here, we calculated the molecular brightness of Sema1b-F254C-mClover to be double that of the molecular brightness of Sema1b-mClover consistent with Sema1b-mClover molecules being present on the membrane of COS-7 cells as monomers (Fig EV1A and B). Click here to expand this figure. Figure EV1. Sema1b is a monomer on the cell surface A. Number and Brightness analysis was used to determine the molecular brightness (ε) of Sema1b-mClover or Sema1b-F254C-mClover in live COS-7 cells. Sema1b seems to be a monomer on the cell surface as the average ε value for Sema1b-mClover (εav = 0.033) is half of the dimeric Sema1b-F254C-mClover (εav = 0.061). The box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, and P-value was calculated by one-way analysis of variance (ANOVA). B. Representative images of COS-7 cells transiently expressing Sema1b-mClover or Sema1b-F254C-mClover in the Number and Brightness experiment. The average intensity images (first column from the left, grey colour) are shown along with the brightness images (second column from the left, rainbow pseudocolor). Scale bar, 40 μm. C. Number and Brightness analysis shows that Sema1becto fails to dimerise PlexA-mClover on the cell surface of COS-7 cells contrary to the dimeric Sema1becto-F254C. The PlexA-mClover construct contained the ectodomain followed by a transmembrane segment and the C-terminal fluorescent protein mClover. The box limits indicate the 25th and 75th percentiles, centred lines show the median, squares represent sample means, whiskers extend 1.5-fold the interquartile range from the 25th and 75th percentiles, and P-value was calculated by one-way analysis of variance (ANOVA). D. Dimerisation of PlexA analysed by Number and Brightness assay. Representative images of COS-7 cells transiently expressing PlexA-mClover before and after treatment with monomeric Sema1becto or dimeric Sema1becto-F254C. The average intensity images (first and second column from the left, grey colour) are shown along with the brightness images (third and fourth column from the left, rainbow pseudocolor). Scale bar, 40 μm. E. Size exclusion chromatography with multi-angle light scattering of PlexA1-4 showing the molar mass and elution profiles at three initial PlexA1-4 concentrations of 2.0 (red), 1.0 (blue) and 0.5 mg/ml (green). Experimental mass of 75 kDa indicates that PlexA1-4 is a monomer. F. Sedimentation coefficient distribution of PlexA1-4 determined by sedimentation velocity analytical ultracentrifugation at a concentration of 33 μM. Calculated sedimentation coefficient (sw(20,w) = 5.0 S) corresponds to the predicted sedimentation coefficient for the monomer. G. Zoomed-in view of the side-on interface showing three main binding sites. Interacting residues are shown in purple. H. Superposition showing the side-on Sema1b-PlexA (blue-orange) interaction from the 2:2 complex overlayed onto the Drosophila Sema2a dimer (pdb 6qp7) structure (grey). The superposition is based on the Sema1b and Sema2a (chain A) sema domains. The clashes between PlexA and Sema2a (chain B) indicate that the side-on binding mode only becomes possible if a semaphorin is in the monomeric state, because the interaction site is otherwise occluded by the dimer interface. Download figure Download PowerPoint Our previous studies have shown that although monomeric, Sema1becto maintains PlexA binding in the nanomolar range (Rozbesky et al, 2019). To investigate whether Sema1becto dimerises or clusters PlexA on live cell surfaces, we probed the molecular brightness of PlexA-mClover on the membrane of COS-7 cells before and after stimulation with purified wild-type Sema1becto or the disulphide-linked dimer Sema1becto-F254C. The PlexA-mClover construct contained the ectodomain followed by a transmembrane segment and the C-terminal fluorescent protein mClover. The addition of Sema1becto-F254C resulted in a significant 3.0 ± 1.8 fold increase of the average molecular brightness which is likely related to a change of the PlexA-mClover oligomeric state. Conversely, the addition of wild-type Sema1becto at the same concentration had no noticeable effect on the average molecular brightness (1.2 ± 0.7 fold increase) (Fig EV1C and D). Thus, though Sema1b binds PlexA in the nanomolar range, it fails to mediate PlexA dimerisation on the cell surface, presumably due to its monomeric state. A novel binding mode revealed by the crystal structure of the PlexA-Sema1b complex We next determined crystal structures of the PlexA1-4-Sema1b1-2 complex from two different crystal forms (1:1 complex and 2:2 complex) at 3.0 and 4.8 Å resolution (Fig 1A–C, Table 1). The 1:1 complex crystal lattice contains one PlexA1-4 monomer and one Sema1b1-2 monomer per asymmetric unit. The crystal packing provides no Sema1b1-2 dimerisation resembling that of the generic homodimeric architecture. The Sema1b1-2 bound in the 1:1 complex with PlexA1-4 is very similar to the unbound Sema1b1-2 with the Cα rmsd of 0.81 Å indicating no large conformational changes upon complex formation. Only small differences in loop orientations at the ligand–receptor interface are apparent. The ectodomain of Drosophila PlexA has not been structurally characterised previously. The PlexA1-4 structure in the 1:1 complex contains a sema domain composed of a seven-bladed β-propeller fold, which is followed by a PSI domain; however, we were not able to locate the IPT1-PSI2 domain segment. The sema domain of PlexA is most similar to mouse PlxnA2 with an rmsd of 1.43 Å over 424 matched Cα positions. In the 1:1 complex crystal structure, PlexA1-4 and Sema1b1-2 interact through their sema domains in a head-to-head orientation similar to the generic architecture shared by all reported structures of semaphorin–plexin complexes (Janssen et al, 2010; Liu et al, 2010; Nogi et al, 2010). The PlexA1-4-Sema1b1-2 interface buries a total solvent-accessible area of 1,837 Å2. This extensive interface is composed of a mixture of hydrophobic and hydrophilic interactions similar to that of PlxnA2-Sema6A. Figure 1. Sema1b binds PlexA at two independent sites, A and B, indicating two independent Sema1b-PlexA orientations: head-to-head and side-on A. Schematic domain organisation of Drosophila PlexA and Sema1b (SS, signal sequence; TM, transmembrane region, red symbols represent the position of N-linked glycosylation sites). B, C. Ribbon representation of the PlexA1-4-Sema1b1-2 1:1 (B) and 2:2 (C) complexes. N-glycans are shown in stick representation. D. Ribbon representation of the side-on orientation derived from the 2:2 complex. E. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto (red) or Sema1becto-mutA (blue) or Sema1becto-mutB (green,) or Sema1becto-mutA+B (orange). The Sema1becto wild-type binding results are as reported previously (Rozbesky et al, 2019). All data were collected at the same time, and error bars represent s.d. of three technical replicates. Download figure Download PowerPoint Table 1. Data collection and refinement statistics PlexA1-4-Sema1b1-21:1 complex PlexA1-4-Sema1b1-22:2 complex Data collection Space group C 2 2 21 P 65 2 2 Cell dimensions a, b, c (Å) 130.9, 195.1, 124.8 153.6, 153.6, 425.4 α, β, γ (°) 90, 90, 90 90, 90, 120 Resolution (Å) 76.86–2.96 (3.07–2.96) 127–4.80 (4.97–4.80) Unique reflections 31,584 (2,791) 15309 (1,485) Multiplicity 4.6 (2.2) 20.9 (19.8) Completeness (%) 93.78 (83.79) 99.90 (100.00) I/σ(I) 8.86 (1.75) 7.41 (1.19) Wilson B-factor (Å2) 69.89 217.35 R-merge (%) 17.4 (63.4) 33.9 (224.1) CC1/2 0.99 (0.548) 0.998 (0.492) CCa 0.997 (0.841) 1 (0.812) Refinement Resolution (Å) 76.86–2.96 (3.06–2.96) 127–4.80 (4.97–4.80) Reflections used in refinement 31582 15303 Rwork/Rfree (%) 18.62/24.63 28.66/30.68 Number of atoms 8,119 15,728 Protein 7,941 15,350 Ligands 178 378 B-factor (Å2) Protein 73.66 260.8 Ligand 129.39 297.52 R.m.s. deviations Bond lengths (Å) 0.005 0.01 Bond angles (°) 0.76 1.43 Ramachandran Favoured (%) 95.28 95.31 Allowed (%) 4.72 4.69 Outliers (%) 0 0 a Highest resolution shell is shown in parenthesis. In the crystal of 2:2 complex, two pairs of 1:1 complexes are packed together in the asymmetric unit with a relative orientation of 168.8° to form a pseudo tetramer. Each Sema1b molecule binds to both PlexA molecules in the pseudo tetramer. There are therefore two independent interaction sites, A and B (Fig 1C), involving two different Sema1b-PlexA orientations. The first, head-to-head orientation is equivalent to that observed in the 1:1 complex (interaction site A; Fig 1B). In the second interaction mode, termed side-on (Fig 1D), Sema1b1-2 and PlexA1-4 bind through the site B or B' with their carboxy-terminal PSI domains oriented in parallel. The B and B' binding sites are not identical within the pseudo tetramer. While the B interaction site is extensive, the B' interaction site is formed through distant contacts between three residues only. Although the two PlexA molecules in the pseudo tetramer form a substantial interface, we did not observe any propensity for PlexA1-4 to dimerise in solution to a concentration at least of 33 μM (Fig EV1E and F). The structure of individual Sema1b1-2 and PlexA1-4 molecules in the 2:2 complex is very similar to those observed in the 1:1 complex, showing no significant conformational changes with the exception of the Exβ1-β2 loop in the extrusion of Sema1b1-2, which adopts a different orientation in order to avoid steric clashes with PlexA1-4. Unfortunately, we were not able to model the Exβ1-β2 loop completely because of fragmentary electron density; however, the loop's position is consistent with it making interactions to the PlexA1-4. As well as undergoing this large-scale reorientation to accommodate PlexA1-4, the Exβ1-β2 loop may also stabilise the complex. In the side-on orientation, the sema domains of Sema1b1-2 and PlexA1-4 are bound in a configuration in which the bottom face of PlexA1-4 is oriented perpendicularly to the side edge of Sema1b1-2 (Fig 1D). The position of the B binding site between Sema1b and PlexA in the 2:2 complex is different to the binding site of the co-receptor neuropilin in the previously reported mouse Sema3A-PlexinA2-Nrp1 ternary complex (Janssen et al, 2012); however, they are positioned in very close proximity (Fig EV2A–C). Interface B can be divided into three main binding sites (Fig EV1G). The most prominent, site 1, is formed by the extrusion of Sema1b and blade 6 of PlexA. Site 2 is composed of the β4B-β4C loop of Sema1b and the β4D-β5A loop of PlexA. In site 3, the N-linked glycan at residue N289 of Sema1b forms contacts with the PSI1 domain of PlexA. The Exβ1-β2 and β4B-β4C loops are involved in semaphorin homodimerisation (Siebold & Jones, 2013); however, for Sema1b1-2 in the 2:2 complex these loops mediate interaction with PlexA suggesting that a B-type interaction can only be mediated by a monomeric semaphorin molecule (Fig EV1H). Click here to expand this figure. Figure EV2. Sema1b binds PlexA at two independent sites A. Ribbon representation of the PlexA-Sema1b side-on orientation. B. The positioning of Nrp1, which is wedged between the sema domains of semaphorin and plexin, in the Sema3A-PlxnA2-Nrp1 ternary complex (pdb 4gza) in ribbon representation. C. Superposition of (A) and (B) on the basis of the sema domains of Sema1b and Sema3A. Superposition shows that the neuropilin binding site and the interaction site B in the side-on orientation are in very close proximity without any significant steric clashes. D–L. Microscale thermophoresis binding experiment for PlexA1-4-mVenus and Sema1becto-mutA (D-F) or Sema1becto-mutB (G-I) or Sema1becto-mutA+B (J-L). (D, G, J) Capillary scans of PlexA1-4-mVenus in standard capillaries. (E, H, K) Fluorescence time traces recorded by MST instrument. The cold and hot regions used for fitting are shown by blue and red vertical lines, respectively. Close-up views of the hot regions are shown in the middle. (F, I, L) The binding curves of Sema1becto to PlexA1-