Structural Evidence for an Octameric Ring Arrangement of SARM1
Sporny, M., Guez-Haddad, J., Lebendiker, M., Ulisse, V.,Volf, A., Mim, C., Isupov, M.N., Opatowsky, Y.
In some pathological conditions, axonal degeneration does not employ the standard apoptotic pathways, and instead involves the SARM1 protein, which induces axonal degeneration in response to various insults, and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. In this study we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome - well known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM1-2 domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We have mutated several interacting SAM1-2 interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development.
Leukocyte Cytoskeleton Polarization Is Initiated by Plasma Membrane Curvature from Cell Attachment
Ren, C. #, Yuan, Q. #, Braun, M., Zhang, X., Petri, B., Zhang, J., Kim, D., Guez-Haddad, J., Xue, W., Pan, W., Opatowsky Y,
Polleux F, Karatekin E*, Tang W, Wu D*.
# Co-first authors
* Co-corresponding authors
Cell polarization is important for various biological processes. However, its regulation, particularly initiation, is incompletely understood. Here, we investigated mechanisms by which neutrophils break their symmetry and initiate their cytoskeleton polarization from an apolar state in circulation for their extravasation during inflammation. We show here that a local increase in plasma membrane (PM) curvature resulting from cell contact to a surface triggers the initial breakage of the symmetry of an apolar neutrophil and is required for subsequent polarization events induced by chemical stimulation. This local increase in PM curvature recruits SRGAP2 via its F-BAR domain, which in turn activates PI4KA and results in PM PtdIns4P polarization. Polarized PM PtdIns4P is targeted by RPH3A, which directs PIP5K1C90 and subsequent phosphorylated myosin light chain polarization, and this polarization signaling axis regulates neutrophil firm attachment to endothelium. Thus, this study reveals a mechanism for the initiation of cell cytoskeleton polarization.
Structural Principles in Robo Activation and Auto-inhibition
Barak, R. #, Yom-Tov, G. #, Guez-Haddad, J., Gasri-Plotnitsky, L., Maimon, R., Cohen-Berkman, M., McCarthy, A.A., Perlson, E., Henis-Korenblit, S., Isupov, M.N., and Opatowsky, Y.
# Co-first authors
Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from "off" to "on" states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.
Approaching the Roundabout: cis and trans Robo1 Contacts Revealed
Guez-Haddad, J., Yom-Tov, G., and Opatowsky, Y.
How neurons and neuronal processes accurately navigate over long distances through dense biological material is a fascinating question in development, which is also important for the understanding of various neurodevelopmental disorders. Key players in this process are Roundabout (Robo) cell surface receptors, which, in response to extracellular ligand stimulation, re-direct the advancement of neurons and axons. For example, in bilateral creatures, neurons from one side of the body project axons across the midline to form synapses on the other side. Robo receptors, in concert with their Slit ligands, repulse axons that have already crossed the midline, thereby preventing repetitive crossing and re-crossing. Here, we review recent advances in our understanding of Robo structure, and how these relate to Robo function in axon guidance and midline crossing, including the recent EM and high-resolution structures of Robo1.
Robo Ig4 Is a Dimerization Domain
Robo receptors play pivotal roles in axonal guidance as well as in neurogenesis, angiogenesis, cell migration, and cancer progression and invasiveness. They are considered to be attractive drug targets for the treatment of cancer, ocular neovascular disorders, chronic kidney diseases, and more. Despite their great importance, the mechanisms by which Robo receptors switch from their "off" to "on" states remain obscure. One possibility involves a monomer-to-dimer or dimer-to-monomer transition that facilitates the recruitment and activation of enzymatic effectors to instigate intracellular signaling. However, it is not known which domains mediate Robo dimerization, or the structural properties of the dimeric interactions. Here, we identify the extracellular Ig4 (D4) as a Robo dimerization domain. We have determined the crystal structure of the tandem Ig4-5 domains (D4-5) of human Robo2 and found that a hydrophobic surface on D4 mediates close homotypic contacts with a reciprocal D4. Analytical ultracentrifugation measurements of intact and mutated D4-5 shows that dimerization through the D4 interface is specific and has a dimerization dissociation constant of 16.9μM in solution.
Yom-Tov, G., Barak, R., Matalon, O., Barda-Saad, M., Guez-Haddad, J., and Opatowsky, Y.
Structural History of Human SRGAP2 (Slit-Robo-GAP2) Proteins
Sporny, M. #, Guez-Haddad, J. #, Kreusch, A., Shakartzi, S., Neznansky, A., Cross, A., Isupov, M.N., Qualmann, B., Kessels, M.M., and Opatowsky, Y.
# Co-first authors
In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4-2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C.
Molecular Symmetry-Constrained Systematic Search Approach to Structure Solution of the Coiled Coil SRGAP2 F-BARx Domain
Michael Sporny, Julia Guez-Haddad, David G. Waterman, Michail N. Isupov*, and Yarden Opatowsky*
* Co-corresponding authors
The extended F-BAR (F-BARx) domain of SRGAP2 generates membrane protrusions when expressed in COS-7 cells, while most F-BARs induce the opposite effect: membrane invaginations. As a first step to understand this discrepancy, the F-BARx domain of SRGAP2 was isolated and crystallized. Diffraction data were collected from two significantly non-isomorphous crystals in the same monoclinic C2 space group. A correct molecular-replacement solution was obtained by applying a molecular symmetry-constrained systematic search approach that took advantage of the conserved biological symmetry of the F-BAR domains. It is shown that similar approaches can solve other F-BAR structures that were previously determined by experimental phasing. Diffraction data were reprocessed with a high-resolution cutoff of 2.2 Å, chosen using less strict statistical criteria. This has improved the outcome of multi-crystal averaging and other density-modification procedures.
The strength and cooperativity of KIT ectodomain contacts determine normal ligand-dependent stimulation or oncogenic activation in cancer
Reshetnyak AV, Opatowsky Y, Boggon TJ, Folta-Stogniew E, Tome F, Lax I, Schlessinger J.
The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells, and interstitial pacemaker cells. Oncogenic KIT mutations play an important "driver" role in gastrointestinal stromal tumors, acute myeloid leukemias, and melanoma, among other cancers. Here we describe the crystal structure of a recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain, rendering KIT tyrosine kinase activity constitutively activated. The structural analysis, together with biochemical and biophysical experiments and detailed analyses of the activities of a variety of oncogenic KIT mutations, reveals that the strength of homotypic contacts and the cooperativity in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regulating normal ligand-dependent or oncogenic RTK activation via a "zipper-like" mechanism for receptor activation.
The Neuronal Migration Factor srGAP2 Achieves Specificity in Ligand Binding through a Two-Component Molecular Mechanism
Guez-Haddad J, Sporny M, Sasson Y, Gevorkyan-Airapetov L, Lahav-Mankovski N, Margulies D, Radzimanowski J, Opatowsky Y.
srGAP proteins regulate cell migration and morphogenesis by shaping the structure and dynamics of the cytoskeleton and membranes. First discovered as intracellular effectors for the Robo1 axon-guidance receptor, srGAPs were later identified as interacting with several other nuclear and cytoplasmic proteins. In all these cases, the srGAP SH3 domain mediates protein-protein interactions by recognizing a short proline-rich segment on the cognate-binding partner. However, as interactions between the isolated SH3 domain and a selected set of ligands show weak affinity and low specificity, it is not clear how srGAPs are precisely recruited to their signaling sites. Here, we report a two-component molecular mechanism that regulates ligand binding to srGAP2 by on the one hand dramatically tightening their association and on the other, moderately autoinhibiting and restricting binding. Our results allow the design of point mutations for better probing of srGAP2 activities, and may facilitate the identification of new srGAP2 ligands.
The Pseudomonas aeruginosa phosphate transport protein PstS plays a phosphate-independent role in biofilm formation
Neznansky, A., Blus-Kadosh, I., Yerushalmi, G., Banin, E.*, and Opatowsky, Y.*
* Co-corresponding authors
Pseudomonas aeruginosa (PA) is a primary cause of nosocomial infections. A key element in PA pathogenicity is its ability to form biofilms that withstand eradication by antibiotics and the immune system. Biofilm formation is controlled by phosphate signaling and here we provide evidence that PstS has a surprising role in this process. Using X-ray crystallography, we characterized the unique underpinnings of PstS phosphate binding and identified an unusual 15-residue N' loop extension. Structure-based experiments showed that PstS-mediated phosphate uptake and biofilm formation are in fact two distinct functions. This places PstS at a junction that separately controls phosphate sensing and uptake and the ultrastructure organization of bacteria.
Crystal structure of the extracellular juxtamembrane region of Robo1
Barak, R., Lahmi, R., Gevorkyan-Airapetov, L., Levy, E., Tzur, A., and Opatowsky, Y.
Robo receptors play pivotal roles in neurodevelopment, and their deregulation is implicated in several neuropathological conditions and cancers. To date, the mechanism of Robo activation and regulation remains obscure. Here we present the crystal structure of the juxtamembrane (JM) domains of human Robo1. The structure exhibits unexpectedly high backbone similarity to the netrin and RGM binding region of neogenin and DCC, which are functionally related receptors of Robo1. Comparison of these structures reveals a conserved surface that overlaps with a cluster of oncogenic and neuropathological mutations found in all Robo isoforms. The structure also reveals the intricate folding of the JM linker, which points to its role in Robo1 activation. Further experiments with cultured cells demonstrate that exposure or relief of the folded JM linker results in enhanced shedding of the Robo1 ectodomain.
Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers
Opatowsky, Y., Lax, I., Tome, F., Bleichert, F., Unger, V. M., and Schlessinger, J.
Stem cell factor (SCF) mediates its cellular responses by binding to and activating KIT, a transmembrane receptor tyrosine kinase. Here we describe an EM 3D reconstruction of negatively stained preparations of SCF-stimulated full-length KIT dimers. Assessment of the EM structure in respect to X-ray crystal structures of KIT extracellular and tyrosine kinase domains reveals the relative positioning of the individual domains in the context of the entire SCF-KIT complex. Whereas the homotypic contacts between the two KIT protomers show a consistent twofold symmetry for the ectodomains, the cytoplasmic arrangement is asymmetric and is found in several discrete conformations. The observed asymmetric contacts between tyrosine kinases may represent molecular interactions occurring during trans autophosphorylation and kinase stimulation.
Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling
Structural analyses of the extracellular region of stem cell factor (SCF) receptor (also designated KIT) in complex with SCF revealed a sequence motif in a loop in the fourth Ig-like domain (D4) that is responsible for forming homotypic receptor contacts and for ligand-induced KIT activation and cell signaling. An identical motif was identified in the most membrane-proximal seventh Ig-like domain (D7) of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and VEGFR3. In this report we demonstrate that ligand-induced tyrosine autophosphorylation and cell signaling via VEGFR1 or VEGFR2 harboring mutations in critical residues (Arg726 or Asp731) in D7 are strongly impaired. We also describe the crystal structure of D7 of VEGFR2 to a resolution of 2.7 A. The structure of D7 dimer is very similar to the structure of D4 dimers seen in the crystal structure of KIT extracellular region in complex with SCF. The high similarity between VEGFR D7 and KIT D4 in both structure and function provides further evidence for common ancestral origins of type III and type V RTKs. It also reveals a conserved mechanism for RTK activation and a novel target for pharmacological intervention of pathologically activated RTKs.
Yang, Y., Xie, P., Opatowsky, Y., Schlessinger, J.
Surface binding inhibitors of the SCF Kit protein-protein interaction
KIT is a receptor tyrosine kinase (RTK), the interaction of which with its ligand, stem cell factor (SCF), is essential for growth and differentiation of various cells. SCF binding promotes KIT dimerization, transphosphorylation, and activation of downstream cell signaling pathways essential for cell proliferation, differentiation, and survival. Gain-of-function mutations in KIT have been identified in human cancers such as gastrointestinal stromal tumors (GIST).[3,4] It was also demonstrated that autocrine or paracrine mechanisms mediated by aberrant expression of SCF and/or KIT might also lead to oncogenesis.[5–7] Because most cases of GIST are driven by oncogenic KIT mutations resulting in enhanced tyrosine kinase activity, inhibitors of the tyrosine kinase activity of KIT, such as Gleevec® (imatinib mesylate) and Sutent® (sunitinib), have been successfully applied for the treatment of GIST patients.
Margulies, D. #, Opatowsky, Y. #, Fletcher, S., Saraogi, I., Tsou, L.K., Saha, S., Lax, I., Schlessinger, J.*, and Hamilton, A*.
# Co-first authors
* Co-corresponding authors
Structural basis for activation of the receptor tyrosine kinase Kit by stem cell factor
Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4 interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.
Yuzawa, S#., Opatowsky, Y#., Zhang, Z., Mandiyan, V., Lax, I., and Schlessinger, J.
# Co-first authors
Structure–function studies of the G-domain from human gem, a novel small G-protein
Gem, a member of the Rad/Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 A resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.
Opatowsky, Y., Sasson, Y., Shaked, I., Ward, Y., Chomsky-Hecht, O., Litvak, Y., Selinger, Z., Kelly, K., and Hirsch, J.A
Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain
Opatowsky, Y., Chomsky-Hecht, O., Kang, M. G., Campbell, K. P., and Hirsch, J. A
Voltage-dependent calcium channels (VDCC) are multiprotein assemblies that regulate the entry of extracellular calcium into electrically excitable cells and serve as signal transduction centers. The alpha1 subunit forms the membrane pore while the intracellular beta subunit is responsible for trafficking of the channel to the plasma membrane and modulation of its electrophysiological properties. Crystallographic analyses of a beta subunit functional core alone and in complex with a alpha1 interaction domain (AID) peptide, the primary binding site of beta to the alpha1 subunit, reveal that beta represents a novel member of the MAGUK protein family. The findings illustrate how the guanylate kinase fold has been fashioned into a protein-protein interaction module by alteration of one of its substrate sites. Combined results indicate that the AID peptide undergoes a helical transition in binding to beta. We outline the mechanistic implications for understanding the beta subunit's broad regulatory role of the VDCC, particularly via the AID.
The voltage-dependent calcium channel beta subunit contains two stable interacting domains
Opatowsky, Y., Chomsky-Hecht, O., Kang, M. G., Campbell, K. P., and Hirsch, J. A.
Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.