Phenotypic effects of Ehlers-Danlos syndrome-associated mutation on the FnIII domain of tenascin-X

Abstract

Tenascin-X (TNX) is an extracellular matrix (ECM) protein and interacts with a wide variety of molecules in the ECM as well as on the membrane. Deficiency of TNX causes a recessive form of Ehlers-Danlos syndrome (EDS) characterized by hyperelastic and fragile skin, easy bruising, and hypermobile joints. Three point mutations in TNX gene were found to be associated with hypermobility type EDS and one of such mutations is the V1195M mutation at the 7th fibronectin Type III domain (TNXfn7). To help elucidate the underlying molecular mechanism connecting this mutation to EDS, here we combined homology modeling, chemical denaturation, single molecule atomic force microscopy, and molecular dynamics (MD) simulation techniques to investigate the phenotypic effects of V1195M on TNXfn7. We found that the V1195M mutation does not alter the three-dimensional structure of TNXfn7 and had only mild destabilization effects on the thermodynamic and mechanical stability of TNXfn7. However, MD simulations revealed that the mutation V1195M significantly alters the flexibility of the C'E loop of TNXfn7. As loops play important roles in protein-protein and protein-ligand interactions, we hypothesize that the decreased loop flexibility by V1195M mutation may affect the binding of TNX to ECM molecules and thus adversely affect collagen deposition and fibrillogenesis. Our results may provide new insights in understanding the molecular basis for the pathogenesis of V1195M-resulted EDS.

Figure 1

The tertiary structure of V1195M by homology modeling. TNXfn7 has a typical Ig-like β-sandwich structure with the top β-sheet (consisting of A-B-E strands colored in black) packing against the bottom C′-C-F-G β-sheet (colored in gray). The seven β strands are connected by six loops (dark color). Mutation V1195M is highlighted by ball-and-stick representation.

Figure 2

Far-UV CD spectra of TNXfn7 and its mutant V1195M. The CD spectra of TNXfn7 (in black) and V1195M (in gray) are typical of β-sheet proteins, confirming that TNXfn7 and its V1195M are well folded. The CD spectra of wt TNXfn7 and mutant V1195M are almost identical, indicating mutation V1195M does not result in significant structural changes of TNXfn7.

Figure 3

Equilibrium denaturation curves for TNXfn7 (circle) and V1195M (square). The denaturation curve of V1195M shifts toward slightly lower [GdmCl] as compared with that of wt TNXfn7, suggesting that mutation V1195M destabilizes TNXfn7. Fitting the equilibrium unfolding curves (solid lines) with a two-state model measured a decrease of thermodynamic stability of TNXfn7 by ∼0.8 kcal/mol.

Figure 4

Mutation V1195M leads to a slight decrease in the mechanical stability of TNXfn7. A) Typical force-extension curves of (TNXfn7)₈ (in black) and (V1195M)₈ (in gray). Dotted lines correspond to the WLC fits. The mechanical unfolding of mutant V1195M and wt TNXfn7 show virtually identical contour length increment ΔL_c of ∼28 nm. B) Unfolding force histograms for TNXfn7 (in black) and V1195M (in gray). The average unfolding force is 204 ± 29 pN (avg. ± S.D., n = 866) for wt TNXfn7 and is 190 ± 25 pN (n = 923) for V1195M.

Figure 5

The RMSD and B-factors distribution of Cα atoms. (A) The Cα RMSD calculated from 47 ns MD trajectories. Using the minimized structure as the reference, the average Cα RMSD calculated for TNXfn7 and V1195M is 1.89 and 1.66 Å, respectively. The overall trend of the Cα RMSD distribution differs slightly between TNXfn7 and V1195M. (B) The calculated B-factors distribution of Cα atoms from the last 42 ns MD trajectories. The B-factors of Cα atoms reflect the remarkable difference between the loop flexibility of TNXfn7 (in black) and V1195M (in gray). Compared with TNXfn7, V1195M has a lower B-factor ordered regions in loops BC, CC′, C′E, and FG, indicating a significantly reduced flexibility caused by the V1195M mutation.