Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization

Abstract

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.

Keywords: 2,2-Dimethyl-2-silapentane-5-sulfonate; Corneal dystrophy; DSS; EMI; EMILIN-1 domain; FAS1; FAS1-4; GCD; IPTG; LB; LCD; Lysogeny broth; MD; NMR; NMR structure; OD; PDB; Protein Data Bank; Protein aggregation; Protein misfolding diseases; Proteolytic degradation; RDC; RMSD; RMSF; SUMO; TBCD; TGFBI; TGFBIp; Thiel–Behnke corneal dystrophy; Transforming growth factor beta induced protein (TGFBIp); WT; fasciclin 1 domain; fourth FAS1 domain of TGFBIp; granular corneal dystrophy; isopropyl β-d-1-thiogalactopyranoside; lattice corneal dystrophy; molecular dynamics; nuclear magnetic resonance; optical density; residual dipolar couplings; root-mean-square deviation; root-mean-square fluctuation; small ubiquitin-like modifier; transforming growth factor beta induced gene; transforming growth factor beta induced protein; wild-type.

FIGURE 1. Limited proteolysis of the WT and Arg555Trp mutant FAS1-4 domains

(A) SDS-PAGE analyses of the degradation products of the WT and the Arg555Trp mutant FAS1-4 domains following limited proteolysis using increasing ratios of thermolysin (Th):FAS1-4 domain ranging from 1:1000 to 1:1 (w/w). After separation of the degradation products by SDS-PAGE the fragments were characterized by N-terminal sequencing to identify the cleavage sites. Protein bands unique to the WT FAS1-4 domain digestion are marked by an asterisk and a diamond. (B) Sequence of the human WT FAS1-4 domain (Met502–Ala657) used in the study. The extra N-terminal AG residues present in the heterologous domains are shown within brackets. Residue Arg555 substituted in the Arg555Trp mutant FAS1-4 domain is indicated (bold). The P1′ amino acid residues for the potential cleavage sites for thermolysin (L, I, F, V, A, M) are highlighted in red letters and the N-terminus of the band marked with an asterisk in (A) is indicated with an arrowhead. Overall, it can be observed that the degradation of the Arg555Trp mutant FAS1-4 domain is retarded compared to the WT domain (compare bands at the 1:10 and 1:1 ratios for the two domain variants) suggesting less accessibility of the protease to the mutant domain. Edman degradation of all the protein bands in the gel revealed that all except the band marked with an asterisk in (A) had the N-terminus AGMGTV, which corresponds to the native N-terminal of the heterologous domain. The band marked with an asterisk has the N-terminus LLGDA. This corresponds to a proteolytic cleavage between residues Arg557 and Leu558 as highlighted in (B). The C-terminal RGD-sequence (Arg642–Asp644) is shown in bold face.

FIGURE 2. Structures of the WT and Arg555Trp mutant FAS1-4 domains

(A) ¹H^N, backbone ¹⁵N, ¹³C^α, and backbone ¹³C′ chemical shift variations (defined as δ_Arg555Trp – δ_WT). The mutation site, residue 555 is highlighted with a grey line. The numbered secondary structures (α-helices and β-strands) are shown at the top of the chart for reference according to the consensus of the FAS1-4 domain NMR structures determined in this study. The structural elements in the WT FAS1-4 domain structure include helix αL (Val505–Lys510), helix α1 (Phe515–Ala525), helix α2 (Thr528–Leu531), strand β1 (Thr538–Pro542), helix α3 (Asn544–Ala549), helix α3′ (Pro552–Leu558), helix α4 (Ala562–His572), strand β2 (Ile573–Gly574), helix α5 (Ser580–Gly582), strand β3 (Leu586–Lys590), strand β4 (Lys596–Lys602), strand β5 (Val605–Val608), strand β6 (Glu611–Met619), and strand β7 (Val624–Ile628). Only minor differences in the residues constituting the secondary structural elements were observed between the WT and the Arg555Trp mutant FAS1-4 domains. The bottom two panels shows the secondary structures (SS) predicted by TALOS+ for the WT and Arg555Trp. −1 indicate α-helix, while +1 indicate β-sheet. (B) The 10 best NMR structures of the WT FAS1-4 domain (blue) and the Arg555Trp mutant FAS1-4 domain (red) overlaid. Except for small differences in some loops, which may be ascribed to lack of data, the only significant difference is the orientation and position of the α3′-helix. The side-chains of Arg555 and Trp555 are drawn with sticks. (C) Protein topology diagram of the TGFBIp FAS1-4 domain showing the secondary structures using the nomenclature by Clout et al. [45].

FIGURE 3. Structures of helices α1, α3, α3′, and α4 forming the hydrophobic cavity in the WT and Arg555Trp mutant FAS1-4 domains

(A) The WT FAS1-4 domain is shown in grey cartoons. Arg555 is shown in cyan spheres and is pointing away from the hydrophobic cavity consisting of residues Met517, Leu518, Ala521, Phe547, Leu550, Leu558, Leu559, and Leu565 (cyan transparent surface and sticks). (B) The Arg555Trp mutant FAS1-4 domain is shown in grey cartoons. Residue Trp555 is shown in green spheres and is located within the hydrophobic cavity consisting of Met517, Leu518, Ala521, Phe547, Leu550, Leu558, Leu559, and Leu565 (green transparent surface and sticks). (C) Overlay of the WT and the Arg555Trp mutant FAS1-4 domain structures. The WT domain is shown in grey and Arg555 is indicated by cyan spheres. The Arg555Trp mutant domain is illustrated in green with the Trp555 residue shown in green spheres. The structures are overall very similar, however, Arg555 of the WT is pointing towards the solvent while the Trp555 residue in the Arg555Trp mutant domain is packed towards the protein.

FIGURE 4. Local structural changes in the Arg555Trp mutant FAS1-4 domain caused by insertion of the tryptophan side chain into the hydrophobic cavity

(A) The differences in side chain rotation within the cavity lined by helices α1, α3, α3′, and α4. The WT FAS1-4 domain is shown in light grey, while the Arg555Trp mutant domain is shown in dark grey. The cavity consists of the hydrophobic residues: Met517, Leu518, Ala521, Phe547, Leu550, Leu558, Leu559, and Leu565, which are here shown in cyan in the WT structure and in green in the Arg555Trp mutant structure. Arg555 (cyan) in the WT FAS1-4 domain and Trp555 (green) of the Arg555Trp mutant domain are shown in fat sticks. (B) Cartoon representation of the Arg555Trp mutant FAS1-4 domain structure showing the heavy atoms of Trp555 with green sticks, the remainder of the protein structure is coloured from blue through white to green indicating observed ¹H^N chemical changes. Residues Ala500, Gly501, Ala521, and Ile522 and all proline residues are coloured white to indicate missing chemical shift data and the two C-terminal residues are coloured white to avoid focus on chemical shift changes due to the C-terminal Pro634Ala mutation. (C) Observed and predicted (using shAIC [44]) chemical shift change indexes for the FAS1-4 domain showing ρ_obs, ρ_W, and 0.1* ρ_struct (see definitions in the Experimental Procedures section) in red, black, and green, respectively, as a function of the residue number. Due to proline residues, some values are missing. The mutation site, residue 555 is highlighted with a grey line. The secondary structures are shown at the top of the chart for reference.

FIGURE 5. Local dynamics of residue at position 555 in the WT and Arg555Trp mutant FAS1-4 domains

(A) MD atom-wise root-mean-square-fluctuation (RMSF) of Arg555 in the WT system and Trp555 in the Arg555Trp mutant system illustrates that the Trp555 residue of the mutant system rotates during the simulation. From the RMSF heavy atoms of Arg555 of the WT system, it is evident that the two NH nitrogens of the guanidinium group fluctuate the most. For the Trp555 residue of the Arg555Trp mutant FAS1-4 domain the fluctuations are much more extensive. Especially the atoms lining one part of the aromatic ring, namely Cε3, Cζ3, Cζ2, and Cη2 fluctuate substantially. This indicates rotation of the aromatic tryptophan ring within the hydrophobic cavity in the Arg555Trp mutant system during the simulation. Rotation of the aromatic ring of tryptophan allows the indole NH to interact favourable with the solvent while the hydrophobic part of the side chain is closely packed within the hydrophobic cavity formed by helices α1, α3, α3′, and α4. (B) All NOEs between Trp555-Hε1 and methyl groups should be easily identified. However, rotation of the tryptophan side chain will change the interaction pattern of residue Trp555 during simulation and may explain why only two of the four possible NOE (within 4.5 Å) between methyl groups and the Trp555-Hε1 could be identified. This observation is ascribed to local dynamics.

FIGURE 6. Global flexibility of the WT and Arg555Trp mutant FAS1-4 domains

(A) Average C^α RMSF from the three MD simulations of the WT and the Arg555Trp mutant domains are shown by points in blue and red, respectively. The differences (WT minus Arg555Trp) between the averages are shown in green histograms with the standard deviation indicated by grey error bars. (B) The RMSD’s derived from the NMR structures; differences (WT minus Arg555Trp) are shown in green bars. The WT domain (blue) is less structured as compared to the Arg555Trp mutant domain (red), specifically this applies to the α3′ helix. The exception is for the residues 600–610, where the RMSD is higher for the Arg555Trp mutant domain. (C) The transverse relaxation (T₂) difference in green histograms (WT minus Arg555Trp) of the two FAS1-4 domain variants indicates a more compact structure for the Arg555Trp mutant domain (red) compared to the WT domain (blue). In addition, around the mutation site the WT structure have residues with short T₂, indicative of chemical exchange. (D) Predicted S² order parameters obtained by the RCI method. The WT and the Arg555Trp mutant domains are shown as blue and red curves, respectively. The N-terminal part (residues Pro552–Trp555) of helix α3′ is more flexible in the Arg555Trp mutant while the C-terminal region of the helix and the loop between helices α3′ and α4 (residues Leu558–Lys563) are more rigid in the Arg555Trp mutant FAS1-4 domain compared to the WT domain. The mutation site, residue 555 is highlighted with a grey line. The secondary structures are shown at the top of the chart for reference.

FIGURE 7. Electrostatic potential isocontours for the WT and Arg555Trp mutant FAS1-4 domains

The lowest energy NMR structure minimized in a water box and ionized with NaCl was used as input for the APBS calculations of the electrostatic potential isocontours. (A) Overlaid ribbon plots for the WT structure (grey) and the Arg555Trp mutant structure (green), (B) electrostatic potential isocontours for the WT FAS1-4 domain, and (C) electrostatic potential isocontours for the Arg555Trp mutant FAS1-4 domain shown in three different molecular orientations: top (left), bottom (middle), and side (right) views. Red and blue colours in (B, C) represent electrostatic potentials of +1 and −1 kT/e, respectively. From the top view it is clear that translation of the α3′-helix in the more compact Arg555Trp mutant structure (left, panel C) induces a larger connected positively charged surface in the mutant FAS1-4 domain compared to the WT structure (left, panel B).

FIGURE 8. Aggregation of the WT and Arg555Trp mutant FAS1-4 domains

In triplicate experiments the WT and Arg555Trp mutant FAS1-4 domains were incubated for 10 days at 37 °C in 1×PBS containing sodium azide, protease inhibitors, and TFE as indicated. Protein concentrations of the supernatants before and after incubation were measured. The diagram shows the relative amounts of soluble protein. In the presence of 10% TFE significantly more (p<0.05) of the Arg555Trp mutant had precipitated compared to the WT domain (marked with an asterisk). Error bars represent the standard error of mean (SEM).