Structural and Mechanistic Insights into the Latrophilin3-FLRT3 Complex that Mediates Glutamatergic Synapse Development.

Characterization of LPHN3-OLF and FLRT3-LRR in solution(A), Schematic representation of LPHN3 and FLRT3 domain architecture. N- and C- designate N- and C-termini of the proteins. Dotted lines indicate constructs that did not bind, solid lines indicate constructs that bound and solid lines with terminal diamonds indicate constructs used in B. (B), ITC of LPHN3-OLF injected at 100μM into the cell containing the FLRT3-LRR domain at 10μM. (C), SEC of the purified LPHN3-OLF, FLRT3-LRR and their complex using a Superdex 200-10/300GL Column. Top margin shows the column calibration: 1, γ-globulin, 158KDa; 2, Ovalbumin, 44KDa; 3, myoglobin, 17KDa. OLF eluted with an apparent MW of ~30KDa; LRR at ~ 71KDa; the complex at ~90KDa as expected for a 1:1 stoichiometry. Inset, Coomassie blue staining SDS-PAGE of the purified proteins. (D), Sedimentation velocity of LPHN3-OLF, FLRT3-LRR and their complex, at two concentrations. Overlay of the diffusion corrected integral sedimentation coefficient distributions from the van Holde - Weischet analysis shows a single species with homogeneous distribution for LPHN3-OLF and the complex but significant self-association for FLRT-LRR. The frictional ratio of the monomer suggested a slightly non-globular shape and was determined to be 1.53 (1.48 and 1.58 with 95% confidence intervals). The dimer frictional ratio was found to be more elongated at 1.76 (1.74 and 1.78 with 95% confidence intervals). (E), The van Holde - Weischet analysis of the FLRT3-LRR concentrations reveal two distinct but overlapping species indicating a clear monomer-dimer equilibrium.

Crystal structure of LPHN3-OLF in P65 and C2221 crystal forms(A), Cartoon representation of the overall folding LPHN3-OLF, emphasizing its 5-bladed β-propeller folding. View from the “entry face” colored in rainbow mode from blue (N-terminus) to red (C-terminus). The Ca2+ ion, found in the central channel of the β-propeller, is represented in sphere mode. (B), Cα traces of the two superimposed LPHN3-OLF structures in P65 and C2221 crystal forms. The main structural differences are in the loops 316–329, 392–405, and 425–434 (red for the C2221 and blue for the P65 crystal form). The side chain of Tyr323 is represented to highlight a core stabilization of the loop 316–329 in the P65 form (“closed” conformation), versus the C2221 form (“open” conformation). (C), Comparison between the atomic thermal motions of the structure in both crystal forms along the backbone: from blue and thin for the lowest B-factor values to thick and red for the highest ones. The coloring/thickness of these cartoon representations is scaled in the range of 8.49 and 77.18 Å2, the minimal and maximal values of B-factors from both models. (D), Detailed views of solvent accessible surface of cavities and pockets (Cyan) in the P65 and C2221 crystal forms, highlighting an accessibility of the central pore in the C2221 form. (E), Metal ion binding in the central pore of the P65 and C2221 crystal forms and contoured with the 2mFo-DFc electron density map. Both panels summarize the protein residues and water molecules (red spheres) involved in the octahedric coordination of the observed Ca2+ (P65) or Mg2+ (C2221). The relevant inter-atomic distances (Å) are reported (insets). See also Figure S1).

DXMS analysis profiles of LPHN3-OLF, alone or bound to FLRT3-LRR domain(A), deuteration levels of the fragment 316–329 of LPHN3-OLF domain alone. (B), Schematics of the deuteration levels of loop 316–329 to indicate the deuteration over time. (C), Deuteration level of loop 316–329 of LPHN3-OLF in complex with FLRT3-LRR. (D), Difference in deuteration level of loop 316–329 between the free and bound LPHN3-OLF domain. The percentages of deuteration levels of each peptide fragment at various time points are shown as a heat map color-coded from blue (10%) to red (90%), as indicated at the bottom of each map. Each block under the protein sequence represents a peptide segment analyzed at each of the four time points (from top to bottom: 10, 100, 1,000, and 10,000s). Differential deuteration is shown in a color-coded map ranging from blue (−50%) to red (+50%), as indicated at the bottom of the panel. Proline residues and regions with no amide hydrogen exchange data available are colored in gray. (E), Structure of the entire P65 LPHN3-OLF color coded according to the differential deuteration map. Cyan to dark blue colors indicate increasing protection of LPHN3-OLF in complex with FLRT-LRR, orange indicate increased solvent accessibility. See also Figure S2, Figure S3.

Complex between LPHN3-OLF and FLRT3-LRR(A), Left panel, side view of the surface rendering of the LPHN3-OLF/FLRT3-LRR complex. Green, FLRT3-LRR, Orange, LPHN3-OLF. The view highlights the associating surfaces of the structure that is the N-terminal part of the concave surface of FLRT3-LRR and the lack of contact on the C-terminal part of it. Right panel, front view of the surface rendering of the complex to highlight the ~45°clockwise inclination of OLF through its central pore. (B), “Open book view” of the semi-transparent surface highlighting both ribbon diagram and side chains of the residues involved in the association and the main secondary elements. (C), Surface view of LPHN3-OLF (top) and FLRT3-LRR (bottom) highlighting the relative sequence conservation of the binding surface. Blue indicated high conservation and red indicates lowest conservation scores. Red ovals indicate the same binding surface in the different orientations. LPHN3-OLF was aligned with 20 other OLF domains of latrophilins and FLRT3-LRR was aligned with 73 unique sequences from CSI-BLAST.

Analyses of the LPHN3-OLF/FLRT3-LRR association(A), Electrostatic surface of LPHN3-OLF and FLRT3-LRR in “open book view” to emphasize a charge complementarity within the main interfacing area of both partners (green oval) and a repulsive facing area (orange square). (B), Stereoview of the stabilization of the LPHN3-OLF loop 316–329 in the complex. Interfacing LPHN3-OLF and FLRT3-LRR residues are represented in yellow and cyan respectively, and the potential hydrogen bonds are shown as dashed magenta lines. Loop 316–329 (closed conformation) with the Tyr323, Asp332 residues and Ca2+ are in shown in purple. The central pore β-strands β10, β14 and β18 involved in the Ca2+ binding and the β13-β14 connecting loop that lies on top of the 316–329 loop are also shown in orange. (C), Loop 316–329 in the open state (C2221) of LPHN3-OLF from a superimposition with the complex structure. Red and green mashes represent the molecular surfaces of the residues that would display a steric clash. (D–H), Analyses of the binding between LPHN3-OLF/FLRT3-LRR by site-directed mutagenesis on FLRT3-LRR (D), Models showing the overall position of the mutations. (E), Western blot showing the FLRT3-LRR mutants used in this figure and their different migration pattern due to the additional glycosylation. (F), ITC experimental trace of the LPHN3-OLF injected at 100μM into the cell containing the FLRT3-LRR R117T control mutant at 10μM. (G), ITC experimental trace of the LPHN3-OLF injected at 100μM into the cell containing the FLRT3-LRR F160N mutant at 10μM. This mutant has an additional N-linked glycosylation site at the OLF binding surface that prevents the association. (H), ITC experimental trace of the LPHN3-OLF injected at 100uM into the cell containing the FLRT3-LRR F160A mutant at 10μM.

Overall model of the association in the context of the synapse(A), FLRT-LRR alone tends to weakly dimerize and LPHN3-OLF alone has loop 316-329 that is mobile possibly allowing and Ca2+ and Na+ exchange. The high affinity of the association maintains LPHN3-OLF loop 316–329 closed and dissociates the low-affinity homo-dimerization of FLRT3-LRR. (B), LPHN3 is presynaptic and it binds through the OLF domain to the LRR domain of FLRT3, a post-synaptic protein. With the exception of the central O-linked glycosylated stalk domain drawn between the GAIN domain and the OLF domain, the rest of extracellular domain of LPHN3 has been determined using crystallography. FLRT3 FN3 domain is represented as homology model using other FN3 domains as template. Approximate dimensions are shown in Angstrom (Å).