ABSTRACT
Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
Subject(s)
Nerve Tissue Proteins/ultrastructure , Receptors, Peptide/metabolism , Tenascin/metabolism , Animals , Cell Adhesion/physiology , Crystallography, X-Ray/methods , HEK293 Cells , Humans , K562 Cells , Leucine-Rich Repeat Proteins , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/ultrastructure , Membrane Proteins/metabolism , Membrane Proteins/ultrastructure , Mice , Mice, Inbred C57BL/embryology , Nerve Tissue Proteins/metabolism , Neurites/metabolism , Neurogenesis/physiology , Neurons/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Glycoprotein GPIb-IX Complex/ultrastructure , Protein Binding/physiology , Proteins/metabolism , Proteins/ultrastructure , Receptors, Cell Surface/metabolism , Receptors, Peptide/ultrastructure , Synapses/metabolism , Tenascin/ultrastructureABSTRACT
Pioneering bioinformatic analysis using sequence data revealed that teneurins evolved from bacterial tyrosine-aspartate (YD)-repeat protein precursors. Here, we discuss how structures of the C-terminal domain of teneurins, determined using X-ray crystallography and electron microscopy, support the earlier findings on the proteins' ancestry. This chapter describes the structure of the teneurin scaffold with reference to a large family of teneurin-like proteins that are widespread in modern prokaryotes. The central scaffold of modern eukaryotic teneurins is decorated by additional domains typically found in bacteria, which are re-purposed in eukaryotes to generate highly multifunctional receptors. We discuss how alternative splicing contributed to further diversifying teneurin structure and thereby function. This chapter traces the evolution of teneurins from a structural point of view and presents the state-of-the-art of how teneurin function is encoded by its specific structural features.
ABSTRACT
Teneurins are ancient cell-cell adhesion receptors that are vital for brain development and synapse organisation. They originated in early metazoan evolution through a horizontal gene transfer event when a bacterial YD-repeat toxin fused to a eukaryotic receptor. We present X-ray crystallography and cryo-EM structures of two Teneurins, revealing a ~200 kDa extracellular super-fold in which eight sub-domains form an intricate structure centred on a spiralling YD-repeat shell. An alternatively spliced loop, which is implicated in homophilic Teneurin interaction and specificity, is exposed and thus poised for interaction. The N-terminal side of the shell is 'plugged' via a fibronectin-plug domain combination, which defines a new class of YD proteins. Unexpectedly, we find that these proteins are widespread amongst modern bacteria, suggesting early metazoan receptor evolution from a distinct class of proteins, which today includes both bacterial proteins and eukaryotic Teneurins.
Subject(s)
Platelet Glycoprotein GPIb-IX Complex/chemistry , Platelet Glycoprotein GPIb-IX Complex/metabolism , Alternative Splicing/genetics , Alternative Splicing/physiology , Cell Communication/physiology , Cryoelectron Microscopy , Crystallography, X-Ray , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Platelet Glycoprotein GPIb-IX Complex/genetics , Protein Structure, Secondary , Tenascin/chemistry , Tenascin/genetics , Tenascin/metabolismABSTRACT
Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between ß-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.
Subject(s)
Intercellular Signaling Peptides and Proteins/chemistry , Low Density Lipoprotein Receptor-Related Protein-6/chemistry , Membrane Proteins/chemistry , Amino Acid Motifs , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Gene Expression , HEK293 Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger 'super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.
Subject(s)
Membrane Glycoproteins/chemistry , Multiprotein Complexes/chemistry , Receptors, Cell Surface/chemistry , Receptors, G-Protein-Coupled/chemistry , Receptors, Peptide/chemistry , Amino Acid Sequence , Animals , Cell Adhesion , Crystallography, X-Ray , HEK293 Cells , HeLa Cells , Humans , Mass Spectrometry , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice, Knockout , Molecular Dynamics Simulation , Molecular Sequence Data , Multiprotein Complexes/metabolism , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Sequence Homology, Amino Acid , Surface Plasmon ResonanceABSTRACT
Latrophilins, receptors for spider venom α-latrotoxin, are adhesion type G-protein-coupled receptors with emerging functions in synapse development. The N-terminal region binds the endogenous cell adhesion molecule FLRT, a major regulator of cortical and synapse development. We present crystallographic data for the mouse Latrophilin3 lectin and olfactomedin-like (Olf) domains, thereby revealing the Olf ß-propeller fold and conserved calcium-binding site. We locate the FLRT-Latrophilin binding surfaces by a combination of sequence conservation analysis, point mutagenesis, and surface plasmon resonance experiments. In stripe assays, we show that wild-type Latrophilin3 and its high-affinity interactor FLRT2, but not the binding-impaired mutants we generated, promote HeLa cell adhesion. In contrast, cortical neurons expressing endogenous FLRTs are repelled by wild-type Latrophilin3 and not by the binding-impaired mutant. Taken together, we present molecular level insights into Latrophilin structure, its FLRT-binding mechanism, and a role for Latrophilin and FLRT that goes beyond a simply adhesive interaction.
