Stabilization of the spectrin-like domains of nesprin-1α by the evolutionarily conserved "adaptive" domain

Abstract

Nesprins are located at the outer and inner membranes of the nuclear envelope and help link the cytoskeleton to the nucleoskeleton. Nesprin-1α, located at the inner nuclear membrane, binds to A-type lamins and emerin and has homology to spectrin-repeat proteins. However, the mechanical and thermodynamic properties of the spectrin-like repeats (SLRs) of nesprin-1α and the potential structural contributions of the unique central domain were untested. In other spectrin superfamily proteins, tandem spectrin-repeat domains undergo cooperatively coupled folding and unfolding. We hypothesized that the large central domain, which interrupts SLRs and is conserved in other nesprin isoforms, might confer unique structural properties. To test this model we measured the thermal unfolding of nesprin-1α fragments using circular dichroism and dynamic light scattering. The SLRs in nesprin-1α were found to have structural and thermodynamic properties typical of spectrins. The central domain had relatively little secondary structure as an isolated fragment, but significantly stabilized larger SLR-containing molecules by increasing their overall helicity, thermal stability and cooperativity of folding. We suggest this domain, now termed the 'adaptive' domain (AD), also strengthens dimerization and inhibits unfolding. Further engineering of the isolated AD, and AD-containing nesprin molecules, may yield new information about the higher-order association of cooperative protein motifs.

Figure 1

(A) Schematic representation of recombinant fragments of human nesprin-1α used in this work. Each black box indicates a spectrin-like repeat (SLR). The gray box indicates the adaptive domain (AD). Short grey box indicates the KASH domain. Our nesprin-1α polypeptide starts from residue 125 in Genbank accession no. AF444779. The SLRs were predicted by SMART, and the adaptive domain (AD) was named by virtue of its disorder predicted by DisEMBL but ordered structure measured in the full length protein. (B) SDS-PAGE analysis of purified recombinant SLR2-7, SLR2-5, SLR5-7 SLR2-7ΔAD and AD polypeptides.

Figure 2

Representative CD spectra of nesprin-1α polypeptides SLR2-7, SLR2-5, SLR5-7 SLR2-7ΔAD and AD. Spectra were recorded in PBS buffer (pH 7.4) at 37°C and scaled to 10 μg/ml for comparison.

Figure 3

Profiles of the thermal unfolding of nesprin-1α fragments recorded at 222 nm (A–D) and the first order derivatives of the unfolding curves, respectively (E–H), as well as the AD spectra before and after thermal unfolding (I). A and E represent the unfolding of SLR2-5; B and F represent the unfolding of SLR5-7; C and G represent the unfolding of SLR2-7ΔAD; D and H represent the unfolding of SLR2-7. SLR2-5 and SLR2-7ΔAD are shown as open and closed circles, respectively (n = 2); SLR5-7 and SLR2-7 are shown as open and closed circles, respectively (n = 3, but 2 representative samples are shown). The apparent sample to sample variation for fragments SLR5-7 and SLR2-7, compared to other two pure SLR fragments, may be due to the effects of the adaptive domain. The unfolding of AD was maintained in 101 °C for 1 hour to reach equilibrium and the spectra are repeated twice and the average value is plotted. Each trial (n) includes the repeat of protein expression, purification and unfolding; independent repeats from the same preparation were indistinguishable.

Figure 4

Protein size measured by DLS for SLR2-7 and SLR2-7ΔAD as a function of increasing temperature. Protein size was measured at each indicated temperature, and average peak size was plotted; bars indicate standard deviation. Asterisks indicate that the plotted size is the average of two unresolved peaks, interpreted as a mixture of monomers and dimers at that temperature. With increasing temperature each construct changed from single-peak dimers, to presumed mixtures of dimers and monomers (asterisk), to single-peak monomers. SLR2-7ΔAD completely dissociates at near 42°C; SLR2-7 completely dissociates at near 58°C. Dissociated proteins then extended further, prior to denaturing above 54°C (SLR2-7ΔAD) or above 66°C (SLR2-7). (n=2 independent runs).

Figure 5

Evolutionary conservation of the Adaptive Domain in nesprin-1 and nesprin-2. (A) Amino acid sequence of the AD of nesprin-1 and nesprin-2 from human, mouse, Bos taurus, chicken and Zebrafish (residues 558-786 in human nesprin-1 isoform, AF444779). Overlines indicate left and right halves of the putative ‘divided LEM-like domain’. Underlines in human nesprin-1 indicate ‘Hot loops’ (high mobility) predicted by DisEMBL. The V729L mutation (boxed) causes Emery-Dreifuss muscular dystrophy. (B) Schematic diagrams to scale, showing the relative positions of the ‘divided LEM-like domain in nesprin-1α, disordered regions predicted by the ‘loops/coils’ definition (DisEMBL), disordered regions predicted by the ‘hot-loops’ definition (DisEMBL), and secondary possible structure (predicted by PSIPRED). Star highlights residue V729.

Figure 6

Speculative model extending our results for INM-localized nesprin-1α to the ONM-localized nesprin-1 giant isoform. Adaptive domain (AD)-mediated self-folding and stabilization of dimers is predicted to form INM- and ONM-localized “knots” that mechanically secure and balance the formation of NE-spanning LINC complexes with SUN-domain protein dimers.