Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F

  1. Wout Oosterheert
  2. Katerina T Xenaki
  3. Viviana Neviani
  4. Wouter Pos
  5. Sofia Doulkeridou
  6. Jip Manshande
  7. Nicholas M Pearce
  8. Loes MJ Kroon-Batenburg
  9. Martin Lutz
  10. Paul MP van Bergen en Henegouwen
  11. Piet Gros

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Abstract

Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9–EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9–EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9–EWI-F with potential CD9 homo-dimerization at either end provides a “concatenation model” for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

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  1. Version published to 10.26508/lsa.202000883
  2. Review Commons

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    Reply to the reviewers

    We thank the three reviewers for providing insightful critiques on our manuscript.

    Changes to document and comments made are marked e.g. “Reply 1.1” (referring the Reviewer #1 item #1, etc.) as described below.

    *Reviewer #1 *

    I found this study to be very convincing. Prior studies are referenced appropriately, the text is well written and clear, the figures are clear also. In my opinion the paper does not need further experiment.

    __[1.1] __The conclusions are well supported by the data. However, the concatenation model seems very speculative at this point. Also, it does not take into account the dynamics of these molecules.

    __Reply 1.1: __The concatenation model combines the structural data from our manuscript with prior biochemical insights into tetraspanin homodimerization and with scanning-EM data on immunogold-labeled CD81 and CD9 on cells. It is not completely clear to us what reviewer #1 refers to with “the dynamics of these molecules”. The cryo-EM data revealed that CD9 - EWI-F is a dynamic complex with straight and bent conformations, which could account for both circular and linear arrangements of tetraspanin-microdomains in cell membranes through the higher-order oligomerization of stable CD9 - EWI-F tetramers. Moreover, transient CD9 - CD9 interactions likely yield a variable number of complexes present in these concatenated and flexible strings of complexes. Such a concatenation model indeed requires further validation. However, it is consistent with experimental data and, importantly, provides a long-awaited molecular basis for TEM assembly. Although it was not within the scope of the current study, it will be of great interest to further investigate the concatenation model through detailed cell-biology based approaches.

    **Minor comment:**

    __[1.2] __There seems to be a mix up between the two structures in the following sentence p4: "In CD9EC2 - 4C8, the D loop adopts a partially helical conformation and central residue F176 is sandwiched by 4E8 residues W59 of CDR2 and W102 and R105 of CDR3 (Fig. 1D). In the 4C8-bound CD9EC2 structure the tip of the D loop points more outward and the Cα atom of F176"

    __Reply 1.2: __The first sentence indeed mixed up the two structures and wrongfully mentioned CD9EC2 - 4C8 instead of CD9EC2 - 4E8. This has now been updated: “In CD9EC2 - 4E8, the D loop adopts …”

    *Reviewer #2 *

    The paper is well written and the conclusions made are supported by the data presented.

    __[2.1] __The ternary structure is in agreement with that of CD9 in complex with the related EWI-2 published earlier this year by Umeda et al (ref #25). The present work thus adds little structural insights but may be useful in showing that the interaction pattern seen extends to another EWI protein family member.

    __Reply 2.1: __We agree with reviewer #2 that that the CD9 - EWI-F structure presented in our work is similar to the CD9 - EWI-2 structure published recently by Umeda et al. (ref #25). However, as also pointed out by reviewer #1, we believe that the CD9 - EWI-F structure adds new important information to understand the molecular mechanism underlying the assembly of tetraspanin-enriched microdomains. Notably, the different conformations of the CD9 - EWI-F complex observed in the cryo-EM data provide structural biology evidence for the dynamic nature of the interaction between a tetraspanin and a partner protein, which is consistent with a wealth of prior biochemical data. Guided by the distinct shape of the CD9EC2 - 4C8 densities, we were able to distinguish a range of straight to bent conformations of the complex. CD9 regions that represent known tetraspanin homo-dimerization sites, orient away from EWI-F and are available for interactions. Thus, combining our structural data with previous biochemical interaction data allowed for the generation of a long-awaited model for the assembly of tetraspanin-microdomains at the molecular level. We believe that these implications for TEM assembly will stimulate new, innovative research into the molecular principles that govern the function of tetraspanins.

    __[2.2] __As such it may be acceptable for publication. In this case, the authors should improve the quality of Figs. 3D and 4D.

    __Reply 2.2: __Figures 3D and 4D depict raw cryo-electron microscopy images (micrographs). The protein complexes imaged in this study only contain light atoms (H, N, C, O, S). Therefore, the collected micrographs only reveal low-contrast images of protein particles, and, for a typical cryo-EM experiment, it is required to average particles from thousands of micrographs to obtain a 3-dimensional reconstruction. We would like to keep the raw micrographs in figures 3 and 4, as it will aid cryo-EM scientists in judging the quality of the data.

    *Reviewer #3 *

    The work is technically well performed and clearly presented including methodological details. I just have a few minor comments:

    __[3.1] __Page 4 and Figure S1: it is hard to see how a reliable affinity for 4E8 can be obtained from the cell binding data in S1A, as there is no indication of saturation. It would be good to at acknowledge that this is at best a rough estimate. Fortunately the data for this nanobody in purified situation seems solid.

    __Reply 3.1: __The obtained affinities are indeed an ±estimation based on a non-linear regression curve fitting on the measured data, performed in triplicate. The text has been updated and now reads as “4C8 and 4E8 bind to purified, full-length CD9 as well as to endogenous CD9 expressed on HeLa cells with apparent binding affinities in the nanomolar range (Fig. S1A, B, C)”. Next to that, a table stating the calculated KDs has been included as Fig. S1C.

    __[3.2] __Page 6: Does the absence of micellar density for the EWI-F complex indicate flexibility of the extracellular domain relative to the TM? Does this happen because the classification focuses on the highly elongated Ig region?

    __Reply 3.2: __These are indeed plausible assumptions. We observed highly heterogeneous, elongated particles in the micrograph shown in Fig. 3D, indicating inter-domain flexibility. If the alignment software focusses on certain Ig-like domains, other regions of the protein complex will be averaged out. An additional complexity with these elongated particles was to select an appropriate box size for particle picking and particle extraction, because the particles differ greatly in size based on their orientation (fully elongated side-views vs. much smaller top-views). When taken together, the complex of CD9 with full-length EWI-F was unsuitable for high-resolution structure determination; the subsequent strategy using EWI-FΔIg1-5 resulted in globular particles with less flexibility (Fig. 4D), which allowed for a more detailed structural characterization of the complex.

    __ [3.3] __Page 8: "Recently, a cryo-EM density map has been reported..." - please reference here.

    __Reply 3.3: __We added the appropriate reference to the sentence: “Recently, a cryo-EM density map has been reported of CD9 in complex with an EWI-F homolog, EWI-2 (25).”

    __[3.4] __Relatively little is known about how tetraspanins help to organize partner receptors into defined membrane domains, evidence for which has emerged from super-resolution light microscopy. Based on their structural analysis of the CD9-EWI-F complex, including the heterogeneity apparent in the cryo-EM structure, they propose a feasible concatenation model for higher order oligomerization of these complexes in the membrane. Obviously the model will need to be tested rigorously by mutational analysis, particularly the EWI Ig6 interface, but as it stands the paper is a significant contribution to the field of tetraspanins.

    __Reply 3.4: __From the 8.6 Å cryo-EM data, the amino-acid residues that form the EWI-F Ig6 dimer interface can indeed not be distinguished. However, our data on CD9 in complex with full-length EWI-F (Fig. 3E) and previous cross-linking data (André et al. In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface - PMID: 19703604) support that EWI-F forms dimeric assemblies. Regarding the concatenation model, we therefore think that it will be of great interest to establish the putative CD9 - CD9 interactions (identified through biochemical approaches), that would link CD9 - EWI-F tetramers into higher assemblies, in the context of native membranes. However, investigating these transient interactions would require various non-trivial experiments and was therefore not within the scope of the current study.

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    Referee #3

    Evidence, reproducibility and clarity

    This paper describes the structure of the tetraspanin CD9 and its interaction with the single pass protein EWI-F. The variability in the D loop of EC2 and the domain swapping is a useful addition to the limited structural database of these proteins and correlates with the relatively poor sequence conservation of this region. The key message is that dimerization of the single pass protein extracellular region, and interaction of its transmembrane helix with the tetraspanin, produces a heterodimeric structure that may further oligomerize. The authors propose a feasible concatenation model for higher order oligomerization of these complexes in the membrane.

    The work is technically well performed and clearly presented including methodological details. I just have a few minor comments:

    Page 4 and Figure S1: it is hard to see how a reliable affinity for 4E8 can be obtained from the cell binding data in S1A, as there is no indication of saturation. It would be good to at acknowledge that this is at best a rough estimate. Fortunately the data for this nanobody in purified situation seems solid.

    Page 6: Does the absence of micellar density for the EWI-F complex indicate flexibility of the extracellular domain relative to the TM? Does this happen because the classification focuses on the highly elongated Ig region?

    Page 8: "Recently, a cryo-EM density map has been reported..." - please reference here.

    Significance

    Relatively little is known about how tetraspanins help to organize partner receptors into defined membrane domains, evidence for which has emerged from super-resolution light microscopy. Based on their structural analysis of the CD9-EWI-F complex, including the heterogeneity apparent in the cryo-EM structure, they propose a feasible concatenation model for higher order oligomerization of these complexes in the membrane. Obviously the model will need to be tested rigorously by mutational analysis, particularly the EWI Ig6 interface, but as it stands the paper is a significant contribution to the field of tetraspanins.

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    Referee #2

    Evidence, reproducibility and clarity

    In this paper, Dr. Oosterheert and colleagues report the crystal structures of CD9EC2 bound to nanobodies 4C8 and 4E8. The CD9EC2/4C8 structure was useful in determining a low resolution cryo-EM structure of EWI-F in complex with CD9/4C8. The observed sample heterogeneity of this ternary complex was reduced by deleting the n-terminal five Ig domains of EWI-F, yielding a modest maximum global resolution of ~ 8.6 Å. The structural approaches used are standard. The crystallographic and structure refinement statistics are sound as are the cryo-EM image processing. The overall cryo-EM structure of the ternary complex shows a central EWI-F protein dimer flanked by one CD9 molecule on each side. The paper is well written and the conclusions made are supported by the data presented.

    Significance

    The ternary structure is in agreement with that of CD9 in complex with the related EWI-2 published earlier this year by Umeda et al (ref #25). The present work thus adds little structural insights but may be useful in showing that the interaction pattern seen extends to another EWI protein family member. As such it may be acceptable for publication. In this case, the authors should improve the quality of Figs. 3D and 4D.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    In this article, the authors provide new insights into the structure of the tetraspanin CD9. On the one hand, they provide crystal structures of the large extracellular domain of CD9, alone or bound to two nanobodies. The 3 structures are similar and similar to that of CD81, a related tetraspanin, except for a portion of the molecule, the so-called D-domain, showing flexibility of this domain. On the other hand, they obtained the cryo-EM structure of CD9 in association with a known-partner (EWI-F) with a resolution of 8.6A. More precisely, the complex of CD9 and the full-length EWI-F showed heterogeneity which they interpret as a consequence of the flexibility between the six Ig-like domains of EWI-F, precluding high-resolution structure determination. However, they showed that CD9 still interacted with a molecule lacking the 5 most membrane-distal Ig domains of EWI-F, and obtained the structure using this construct and an anti-CD9 nanobody. This structure reveals a hetero-tetrameric arrangement of CD9-EWIF, with a central EWI-F dimer flanked by a CD9 molecule on each side. CD9 and EWI-F interact through their transmembrane domains and the two truncated EWI-F molecules through the remaining Ig domains. Importantly, CD9 and EWI-F do not make contacts in the extracellular region, and CD9 shows a semi-open conformation. The structure also shows different configurations of the complex.

    I found this study to be very convincing. Prior studies are referenced appropriately, the text is well written and clear, the figures are clear also.

    In my opinion the paper does not need further experiment.

    The conclusions are well supported by the data. However, the concatenation model seems very speculative at this point. Also, it does not take into account the dynamics of these molecules.

    Minor comment:

    There seems to be a mix up between the two structures in the following sentence p4: "In CD9EC2 - 4C8, the D loop adopts a partially helical conformation and central residue F176 is sandwiched by 4E8 residues W59 of CDR2 and W102 and R105 of CDR3 (Fig. 1D). In the 4C8-bound CD9EC2 structure the tip of the D loop points more outward and the Cα atom of F176"

    Significance

    Tetraspanins have been shown over the years to play an essential role in various biological functions. Among them, CD9 which is strongly expressed on the oocyte plasma membrane is essential for sperm-egg fusion. However, the mechanisms by which CD9 regulates this fusion process as well as other cell-cell fusion events remain unknown. The elucidation of its structure and of how it interacts with well characterized partner proteins is clearly a major advance in our understanding of the function of this molecule.

    The absence of a structure for tetraspanins has been for a long time a knowledge gap. Following a breakthrough in 2001 with the publication of the crystal structure of the large extracellular domain of CD81 (Kitadokoro et al., EMBO J 2001), it was only recently that the structure of a full length tetraspanin, again that of CD81, was published (Zimmermann et al., Cell 2016). Earlier this year was published the crystal structure of a truncated version of CD9 as well as the cryo-EM structure of CD9 in association with another molecular partner EWI-2 (Umeda et al.,Nature com 2020).

    The present structure adds new important information such as the existence of different conformation in the large extracellular domain of CD9 or the structure of CD9 with another molecular partner. It also highlights the different configurations of the complex. It will be of interest to researchers interested in tetraspanins, in membrane organization as well as researchers interested in the biological processes regulated by CD9, notably sperm-egg fusion.

    My field of expertise concerns tetraspanins. I cannot comment on the technical aspects of the structures.

  6. Version published to 10.1101/2020.06.02.130047v1 on bioRxiv