Direct Binding of the Flexible C-Terminal Segment of Periaxin to β4 Integrin Suggests a Molecular Basis for CMT4F

Abstract

The process of myelination in the nervous system requires a coordinated formation of both transient and stable supramolecular complexes. Myelin-specific proteins play key roles in these assemblies, which may link membranes to each other or connect the myelinating cell cytoskeleton to the extracellular matrix. The myelin protein periaxin is known to play an important role in linking the Schwann cell cytoskeleton to the basal lamina through membrane receptors, such as the dystroglycan complex. Mutations that truncate periaxin from the C terminus cause demyelinating peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease type 4F, indicating a function for the periaxin C-terminal region in myelination. We identified the cytoplasmic domain of β4 integrin as a specific high-affinity binding partner for periaxin. The C-terminal region of periaxin remains unfolded and flexible when bound to the third fibronectin type III domain of β4 integrin. Our data suggest that periaxin is able to link the Schwann cell cytoplasm to the basal lamina through a two-pronged interaction via different membrane protein complexes, which bind close to the N and C terminus of this elongated, flexible molecule.

Keywords: Charcot-Marie-Tooth disease; integrin; myelin; periaxin; protein structural & functional analysis.

Figure 1

Identification of an interaction between periaxin and β4 integrin. (A) Top: structure of full-length L-PRX and the bait used in a yeast two-hybrid screen using the C terminus of L-PRX (aa 681–1383) fused to the GAL4 DNA-binding domain. Bottom: structure of the intracellular domain of β4 integrin and one (62B) of the three clones recovered from the screen, all of which included the third FNIII domain of β4 integrin. (B) Identification of the region of PRX, which binds to β4 integrin. The strength of interaction between the 62B clone and a series of PRX constructs in a yeast two-hybrid β-galactosidase assay was assessed semiquantitatively by the time taken for colonies to turn blue (+++, 30 min; ++, 30–60 min; +, 60–180 min). (C) Interaction of β4-FNIII-3 with PRX in vitro. GST or a GST-β4-FNIII-3 fusion protein were incubated with a sciatic nerve lysate in vitro and any bound L-PRX was detected by Western blotting (IB) using a PRX antibody after SDS-PAGE. The input lane confirms the presence of PRX in the lysate. Coomassie blue staining shows that equivalent amounts of GST and the GST fusion proteins were used. (D) Immunoaffinity copurification of β4 integrin and PRX. Detergent extracts of mouse sciatic nerve in the non-ionic detergent Igepal were incubated with beads, to which affinity-purified sheep anti-PRX antibodies had been covalently coupled. After extensive washing, bound proteins were eluted and analyzed by SDS-PAGE and Western blotting. The SDS control lane contains proteins that bound to the beads after first solubilizing the nerves in SDS to disrupt protein-protein interactions, followed by dilution of the SDS with Triton X-100. (E) Coimmunoprecipitation of β4 integrin and PRX from transfected HEK293 cells. PRX and β4 integrin were detected by Western blot (IB) after immunoprecipitation (IP) with β4 integrin antibodies when β4 integrin, α6 integrin, and PRX were coexpressed. Preimmune serum (PI) did not precipitate either protein. Reciprocally, β4 integrin and PRX were detected by Western blot after IP with anti-PRX antibodies, when β4 integrin, α6 integrin, and PRX were coexpressed. (F) Immunohistochemistry. Both PRX (green) and β4 integrin (red) localize at the abaxonal membrane in mature myelin. (G) Quantification of β4 integrin from the IP of crosslinked sciatic nerves from the full-length PRX transgenic mouse on a PRX null background (n = 3) vs. C-terminally truncated PRX transgene on a PRX null background (n = 3).

Figure 2

Direct molecular interaction assays using purified recombinant proteins. (A) Pulldown with pure recombinant proteins on a Ni-NTA affinity matrix. Top, PRX-C and His-tagged β4-FNIII-3; middle, PRX-C alone; bottom; partially degraded PRX-C and His-tagged β4-FNIII-3. Samples: 1, input sample; 2, unbound fraction; 3–5, washes; 6, elution. PRX-C is indicated in red and β4-FNIII-3 in blue. Sizes of molecular weight markers are indicated on the left (kDa). (B) Sequence of the PRX-C construct, indicating the presence of acidic and basic repeats. The underlined segment (the acidic domain) can be detected in all the PRX-C bands in the bottom panel of (A). (C) Size exclusion chromatography (SEC)-MALS analysis shows molecular mass expected for a 1:1 complex. PRX-C, red; β4-FNIII-3, blue; complex, black. (D) Isothermal titration calorimetry (ITC) titration of the complex indicates 1:1 stoichimetry. ΔH = 6.2 ± 0.06 kcal/mol, ΔS = 5.8 cal/mol°. (E) Covalent crosslinking. The complex is only visible when both proteins have been activated (Lane 1). The magenta arrowhead indicates the 1:1 complex. PRX-C is indicated in red and β4-FNIII-3 in blue. Samples: 1, both proteins activated; 2, PRX-C activated; 3, PRX-C activated, β4-FNIII-3 added; 4, no activation; 5, no activation, PRX-C added; 6, no activation, β4-FNIII-3 added; 7, β4-FNIII-3 activated; 8, β4-FNIII-3 activated, PRX-C added.

Figure 3

L-PRX remains disordered in the complex. (A) Synchrotron radiation circular dichroism (SRCD) spectroscopy. PRX-C, red; β4-FNIII-3, blue; complex, black; sum of individual protein spectra, magenta. (B) Secondary structure predictions of the PRX-C acidic region, aligned from selected species.

Figure 4

Crystal structure of rat β4-FNIII-3 (stereo view). Two of the four individual chains in the asymmetric unit are shown. The chain is colored from the N (blue) to the C (red) terminus. The His-tag is partially resolved at the N terminus, and takes different conformations in different monomers. Secondary structure elements are labeled.

Figure 5

Structure of the complex in solution. (A) Small-angle X-ray scattering (SAXS) scattering curve. PRX-C, red; β4-FNIII-3, blue; complex, black. (B) Dimensionless Kratky plot. The black cross indicates the theoretical peak position for a folded, perfectly globular protein. (C) Distance distribution function. (D) Multi-phase model from MONSA. PRX-C, pink; β4-FNIII-3, blue. (E) Rigid-body fit between a chain-like model of PRX-C (pink) and the integrin crystal structure (blue). (F) EOM analysis for PRX-C. Dashed lines, theoretical distribution for a random coil; solid lines, the ensemble for PRX-C. Maximum dimension is shown in red and radius of gyration in black. PRX-C is slightly more elongated than a random coil.

Figure 6

Possible binding site for L-PRX. (A) Surface of the β4-FNIII-3 crystal structure, colored by electrostatic potential. The predicted binding site is shown by a green dashed line. (B) MD simulation indicates the possible binding site is flexible (arrow) and may open up more. (C) RMSF during the 550-ns simulation. (D) Comparison of the start (magenta) and end (yellow/green) points of the simulation. The loop at the potential binding site (arrow) is the most mobile region. The largest peak in the RMSF plot (panel C) is colored green in the post-simulation structure.

Figure 7

Schematic view of the PRX-linked protein scaffold in peripheral nervous system (PNS) myelin. (A) Cross section of a myelinated peripheral axon, showing compact myelin, the abaxonal membrane, Cajal bands, and appositions. While dystrophin-related protein 2 (DRP2) is restricted to the appositions, and PRX is concentrated in them, β4 integrin is involved in signaling in the Cajal bands (Salzer, 2015). (B) A possible architecture for L-PRX-based protein scaffolds at the Schwann cell abaxonal membrane. The position of the truncating R1070X mutation in L-PRX is indicated, and the β4 integrin binding site lies C-terminal to it, although an exact binding site remains to be identified.