Solution NMR backbone assignment of the SASH1 SLy proteins associated disordered region (SPIDER)

Abstract

SASH1 is a scaffold protein with context-dependent biological functions in cell adhesion, tumor metastasis, lung development, and pigmentation. As a member of the SLy protein family, it contains the conserved SLY, SH3, and SAM domains. The 19 kDa SLY domain harbors over 70% of the SASH1 variants associated with pigmentation disorders. However, its solution structure or dynamics have not been investigated yet, and its exact position in the sequence is not clearly defined. Based on the bioinformatic and experimental evidence, we propose renaming this region to the SLy Proteins Associated Disordered Region (SPIDER) and defining the exact position to be amino acids 400–554 of SASH1. We have previously identified a variant in this region linked to a pigmentation disorder, S519N. Here, we used a novel deuteration technique, a suite of TROSY-based 3D NMR experiments, and a high-quality HNN to obtain near complete solution backbone assignment of SASH1’s SPIDER. A comparison with the chemical shifts of non-variant (S519) SPIDER shows that the S519N substitution does not alter the free form solution structural propensities of SPIDER. This assignment is the first step to characterize the role of SPIDER in SASH1-mediated cellular functions and provides a model for the future study of sister SPIDER domains in the SLy protein family.

Biological context

SASH1 is a member of the SLy scaffold protein family and is important in many cellular and developmental functions, including cytoskeletal reorganization (Martini et al. 2011), regulation of cell migration and tumor metastasis (Martini et al. 2011; Lin et al. 2012; Franke et al. 2019; Chen et al. 2020), atherosclerosis (Weidmann et al. 2015), human skin pigmentation(Zhou et al. 2013, 2017; Courcet et al. 2015; Shellman et al. 2015; Wang et al. 2017; Zhong et al. 2019; Cui et al. 2020; Araki et al. 2021; Jaufmann et al. 2021), and mouse lung development (Coulombe et al. 2019). Downregulation of SASH1 expression is a significant negative prognostic, predictive factor in colon, breast, and liver cancer (Zeller et al. 2003; Martini et al. 2011; Jaufmann et al. 2021). Additionally, 19 variants are known to be linked to pigmentation disorders (Zhou et al. 2013, 2017; Courcet et al. 2015; Shellman et al. 2015; Wang et al. 2017; Zhong et al. 2019; Cui et al. 2020; Araki et al. 2021; Jaufmann et al. 2021) (Fig. 1). Despite these multiple critical biological functions, to date only one peer-reviewed structural study has been published(Clements et al. 2022) and two structural data sets have been deposited in the PDB (2EBP and 2DL0) regarding the structural and biochemical properties of SASH1. The SLy family consists of three members: SASH1/SLy3, SASH3/SLy1 and SAMSN1/SLy2 (Jaufmann et al. 2021). All three contain a conserved tri-domain region, which consists of a “SLY”, SH3, and SAM domain (Zhong et al. 2019; Araki et al. 2021; Jaufmann et al. 2021) (Fig. 1). Note that SLy is used to refer to a domain and a protein, which is confusing. This “SLY” domain/region is conserved and predicted to be a disordered region. In SASH1, the “SLY” region has important functions in regulating cell migration and it harbors multiple variants that are associated with pigmentation disorders (Martini et al. 2011; Weidmann et al. 2015; Araki et al. 2021; Jaufmann et al. 2021). However, this domain/region has not been well defined. In this study, we provide bioinformatic evidence and rationale to propose renaming this conserved region to the “SLy Proteins Associated Disordered Region” (SPIDER) and defining the exact position to be amino acids 400–554 of SASH1. This is a unique acronym and is not found in any of the major gene or protein databases. In addition, we present the near complete ¹H-¹⁵N backbone assignment for the S519N variant of SPIDER in SASH1, which we previously reported as associated with a lentiginous pigmentation disorder (Shellman et al. 2015). These data constitute the first step in defining the biochemical role of these regions across the SLy protein family and will provide the basis for the future study of SPIDERs across the SLy protein family.

Fig. 1.

Domain architecture of the SLy protein family showing the conserved SLy Proteins Associated Disordered Regions (SPIDER; formally known as SLY domain), SH3, and SAM domains. The 19 known substitution variants associated with pigmentation disorders are listed with arrows displaying the domain location across the protein. Over 70% of them are in SPIDER/“SLY”. S519N, the variant of interest in this assignment, is shown in red

Methods and experiments

Bioinformatic analyses and definition of region under study

SPIDER is a conserved region in all three members of the SLy family and spans residues 400–554 in SASH1. A Protein BLAST of this region in the family members revealed homologous regions with a sequence identity between 28 and 35% in SAMSN1 (20–163) and SASH3 (20–173) (Fig. 2). However, the literature referencing this region as the “SLY” domain has conflicting and ambiguous definitions. The current definition in the InterPro databases (PF12485) is unclear and contradictory as to whether “SLY” is a domain or full protein (as of March 9, 2023). Specifically, InterPro describes SLY as a eukaryotic domain family from 144 to 156 amino acids long, containing an LGKK motif, and being found associated with an SH3 and SAM domain. It then states that “SLY contains an [SH3] domain and [SAM]”. For future studies of this region and these proteins, an unambiguous consensus on its name and definition needs to be reached. We propose to refer to this region as “SLy Proteins Associated Disordered Region” (SPIDER). This name is unique and not associated with any other protein family or acronyms, and it signifies the family that contains this region and its disordered characteristics. We have conducted sequence alignment of all three family members (Fig. 2) and the adjacent SH3 domain. Based on these data, we propose that spider be defined as spanning residues 400–554 in SASH1, 20–163 in SAMSN1, and 20–173 in SASH3. Specifically, the starting point (residue 400 in SASH1) marks the beginning of the conserved sequence of this region, while the end point (residue 554 in SASH1) is a proline, which is abundant in disordered regions. Additionally, residue 555 marks the start of the SH3 domain as reported in PDB: 2EBP. Based on our analysis here (Fig. 2) and findings by others, we propose SPIDERs should be defined as a conserved sequence spanning 143 to 154 amino acids in length, containing a nuclear localization sequence (NLS) (Uchida et al. 2001; Beer et al. 2005; Franke et al. 2020; Jaufmann et al. 2021), and a conserved LGKK motif of unknown function. Thus, SPIDER of SASH1 spans residues 400–554; SAMSN1-SPIDER spans 20–163, and SASH3-SPIDER spans 20–173.

Fig. 2.

Sequence alignment between SPIDERs of the three members of the SLy protein family, which span the conserved disordered region immediately N-terminal of the SH3 domains. These regions are only present in the SLy/SASH1 family of scaffolding proteins and exhibit 28–35% sequence similarity. Residue colors indicate side-chain chemistry: yellow, proline; blue, hydrophobic; red, positively charged; magenta, negatively charged; green, polar uncharged; orange, glycine; cyan, aromatic

Protein expression and purification

We designed a construct that comprises residues 396–564 in SASH1. This covers the SASH1 SPIDER (400–554) and the N-terminus of the SH3 domain (555–564). The construct which has a substitution at position 519 (S519N) was cloned into the expression vector pET-28a(+) with an N-terminal His-tag coupled to a thrombin cleavage site. The plasmid was transformed into E. coli strain BL21 (LEMO21-DE3). The expression of the triple labeled protein was conducted using the protocol published recently (Li and Byrd 2022) to ensure proper bacterial adaptation to the deuterated minimal media. The deuterated M9 media was formulated with 1 g/L ¹⁵N-ammonium chloride and 2 g/L ¹³C-7D-glucose. The culture was induced with 0.4 mM isopropyl-1-thio-d-galactopyranoside once the A₆₀₀ reached 0.7 and shaken overnight at 20 °C. Cells were harvested by centrifugation at 4 °C for 10 min at 5000 × g. The cells were lysed using low-imidazole binding buffer (20 mM HEPES, 200 mM NaCl, 1 mM EGTA, 1 mM MgCl₂, 1 mM N aN₃, 20 mM imidazole, pH 7.3), disrupted by sonication and cleared by centrifugation at 30,900×g. The lysate was purified using a HisTrap FF column (Cytiva), where the protein was eluted using high-imidazole buffer (20 mM HEPES, 200 mM NaCl, 1 mM EGTA, 1 mM MgCl₂, 1 mM NaN₃, 200 mM imidazole, pH 7.3). The eluted protein was concentrated to a 4 ml volume. Further purification was completed on a size-exclusion HiLoad 16/600 Superdex 75 pg (Cytiva) in NMR buffer (20 mM NaP, 100 mM NaCl, 1 mM DTT, 0.02 % N aN₃, pH 7.0). The relevant fractions were concentrated using a 3000 MWCO concentrator to a concentration of 800 μM before NMR measurements.

NMR spectroscopy

²H–,¹³C–, and ¹⁵N-labeled NMR sample of SPIDER was prepared in the NMR buffer at a concentration of 800 μM and measured in a 5 mm Shigemi tube. The spectra were recorded on BRUKER Avance NEO 600 MHz triple-resonance cryoprobe spectrometers at 5 °C. Backbone assignment was achieved using ¹H-¹⁵N HSQC, 3D HNCACB–, HN(co) CACB–, HNCO–, and HN(ca)CO-TROSY (Ikura et al. 1990; Cavanagh 2007), and HNN (Weisemann et al. 1993). The 3D spectra were acquired with a nonuniform sampling (NUS) scheme generated by the NUS@HMS scheme generator (Hyberts et al. 2012) employing 2048 complex data points in the direct dimension and 25–30% sampling of the original 256 and 92 points in the indirect ¹³C and ¹⁵N dimensions, respectively. For all experiments, the spectral widths were 8196 Hz (¹H), 2129 Hz (¹⁵N), 2113 Hz (¹³C=O), and 12076 Hz (¹³Cα/¹³Cβ), the number of scans was 16 and 32 for 3D experiments and 2D HSQC, respectively, and the interscan delay was 1.0 s. The 3D NUS spectra were reconstructed using the hmsIST software (Hyberts et al. 2012), and the linearly acquired 2D spectra were subject to NUS zero-filling as an alternative to linear prediction. A solvent subtraction function was applied in the direct dimension. Further data processing and visualization were performed using NMRpipe/NMRDraw (Delaglio et al. 1995) and NMRFAM Sparky (Lee et al. 2015). Resonance assignment was performed using the CCPNmr analysis software v 2.5.1 (Vranken et al. 2005).

Extent of assignment and data deposition

The ¹H-¹⁵N HSQC of the SPIDER showed a narrow dispersion of peaks characteristic of a disordered protein (Fig. 3), confirming the prediction. Due to the relatively large protein size, disordered nature and the presence of multiple redundant charged regions, severe peak overlap only enabled us to assign ~ 50% of the backbone without deuteration. Pursuing protein deuteration following a recently introduced optimized protocol (Li and Byrd 2022) in combination with TROSY versions of the 3D backbone assignment experiments supported by a high-quality HNN experiment yielded a signal separation that allowed us to obtain near-to-complete backbone assignment (97.4%) (Table 1; Fig. 3). Of note, lowering the temperature to 5 °C improved the line shapes significantly. The backbone assignment for SPIDER has been deposited in the BMRB with accession code 51747.

Fig. 3.

Deuteration combined with 3D TROSY and HNN experiments allowed for the assignment of 97.4% of the S519N variant of SPIDER. Left: F3-F2 plane showing ¹H–¹⁵N correlations through the 3D HNN spectrum at the F1–¹⁵N chemical shift of 123.2 ppm (orange) overlaid with the 2D ¹H–¹⁵N HSQC spectrum (teal). Off-diagonal peaks (marked with squares and connected by a line) indicate the ¹⁵N chemical shifts of the residues preceding and succeeding the residue represented by the diagonal peak (marked with a circle; L418). Right: ¹H–¹⁵N HSQC spectrum illustrating peak assignment.

Table 1.

Backbone assignment statistics of SPIDER

Construct	Total number of relevant residues*	Total number of relevant non-proline residues	% Backbone resonances assigned (number of backbone atoms assigned)
SASH1 396–564 (Linker: 396–399; SPIDER: 400–554; SH3: 555–564)	169	158	97.4% (153 15N, 161 Cα, 143 Cβ, 156 CO)

^*The construct has 14 additional non-relevant residues from cloning (thrombin cleavage site; His-tag).

Chemical shift analysis

An overlay of ¹H–¹⁵N HSQC spectra of the S519N variant with the wild-type indicates that while minor peak shifts occur, the overall fingerprint remains unchanged. This indicates that the mutation does not alter the protein’s overall structural and dynamic properties (Fig. 4). Thus, the assignment can also be used for wild-type constructs in future studies of SASH1-SPIDER.

Fig. 4.

Overlay of ¹H–¹⁵N HSQC spectra of ¹⁵N-labeled wild-type (W.T.) SPIDER and S519N variant. While minor peak shifts occur, the overall fingerprint remains mostly unchanged, indicating that the mutation preserves the structural and dynamic properties. Both S519 (unpublished data) and N519 are marked.

To assess the presence of residual secondary structure, we computed the residue-specific Secondary Structure Propensity (SSP) score (Marsh et al. 2006) from the HN, N, Cα, Cβ, and CO chemical shifts (Fig. 5). The SSP score ranges from +1 for a fully formed helix to −1 for a fully formed β-sheet and is ~ 0 for loops and disordered residues. Despite of the overall disordered nature of SPIDER, there are many regions with some β-strand propensity separated by more disordered linkers. Considering SASH1’s established role as a scaffolding protein, an intriguing avenue for further investigation is whether these strands act as nucleation sites for oligomerization events or participate in specific binding interactions. Notably, residue 519 is situated at the N-terminus of one of these transient β-strands (519–529), potentially linking the mutation to disorders associated with this domain.

Fig. 5.

Secondary structure propensities derived from chemical shift values of ¹HN, ¹⁵N, Cα, Cβ, and CO. An SSP score of +1 indicates a fully formed α-helix, while −1 indicates a fully formed β-sheet. Multiple regions show some strand propensity and are separated by more disordered parts.

Data availability

The chemical shift assignment of the S519N variant of SPIDER (BMRB 51747) has been deposited in the Biological Magnetic Resonance Data Bank.