Interdomain tilt angle determines integrin-dependent function of the ninth and tenth FIII domains of human fibronectin

Abstract

Integrins are an important family of signaling receptors that mediate diverse cellular processes. The binding of the abundant extracellular matrix ligand fibronectin to integrins alpha(5)beta(1) and alpha(v)beta(3) is known to depend upon the Arg-Gly-Asp (RGD) motif on the tenth fibronectin FIII domain. The adjacent ninth FIII domain provides a synergistic effect on RGD-mediated integrin alpha(5)beta(1) binding and downstream function. The precise molecular basis of this synergy remains elusive. Here we have dissected further the function of FIII9 in integrin binding by analyzing the biological activity of the FIII9-10 interdomain interface variants and by determining their structural and dynamic properties in solution. We demonstrate that the contribution of FIII9 to both alpha(5)beta(1) and alpha(v)beta(3) binding and downstream function critically depends upon the interdomain tilt between the FIII9 and FIII10 domains. Our data suggest that modulation of integrin binding by FIII9 may arise in part from its steric properties that determine accessibility of the RGD motif. These findings have wider implications for mechanisms of integrin-ligand binding in the physiological context.

Fig. 1

Ribbon diagram of the FIII9–10 domain pair. Atomic coordinates were obtained from the Protein Data Bank (43) (Protein Data Bank code 1FNF) and imaged using RasTop (available on the World Wide Web at www.openrasmol.org). Residues substituted in this study, shown in their putative mutant conformation as rendered by Swiss PDB Viewer (available on the World Wide Web at spdbv.niehs.nih.gov), are displayed in ball-and-stick format and labeled accordingly using one-letter code. The dotted line designates disulfide bond present in oxidizing conditions. The synergistic PHSRN sequence and the RGD motif are indicated in yellow and blue, respectively.

Fig. 2

Thermodynamic stability of FIII9–10 proteins. a, normalized chemical denaturation curves for FIII9 unfolding, corresponding to the first transition region of the consecutive unfolding of FIII9 and FIII10 within wild type and mutant FIII9–10 variants, as depicted in the inset. Open squares, FIII9–10; filled squares, FIII9′10; blue circles, FIII9′10-CC_red; red circles, FIII9′10-CC_ox. Denaturation data for FIII9′10-CC_ox are presented across the entire denaturation range encompassing both domains, since they unfold cooperatively. b, comparison of [GdnHCl]_½ values, derived from regression analysis of the curves in a, for wild type (wt) FIII9–10, FIII9′10, FIII9′10-CC_red, and FIII9′10-CC_ox (left to right).

Fig 3

Effect of disulfide linkage on the biological activity of FIII9–10. a, attachment of BHK cells on surface-immobilized FIII9–10 variants and a single FIII10 domain. Results are expressed relative to the maximum level of attachment attained at higher concentrations of FIII9′10 (not shown). b, assessment of the ability of the FIII9–10 variants to support BHK cell spreading. c and e, normalized dose-response data for solid-phase binding of FIII proteins to surface-bound integrin α₅β₁ (c) or α_vβ₃ (e). Open squares, FIII9–10; filled squares, FIII9′10; blue circles, FIII9′10-CC_red; red circles, FIII9′10-CC_ox; open triangles, FIII10. d and f, apparent K_d values derived from the curves in c and e, for binding of FIII9′10, FIII9′10-CC_red, FIII9′10-CC_ox, and FIII10 (left to right) to integrin α₅β₁ (d) or α_vβ₃ (f).

Fig 4

Combined ¹⁵N and ¹H chemical shift differences, Δδ, between FIII9′10 and FIII9′10-CC_red (a) and FIII9′10 and FIII9′10-CC_ox (b). Δδ is given by Δδ = √((δ¹H)² + (δ¹⁵N/6)²). Values greater than 0.08, indicated by the horizontal dotted line, are considered to be significant. The filled horizontal bars denote the location of β-strands, and the vertical dotted lines mark the sites of the mutated residues Ala¹³⁴⁰ and Val¹⁴⁴². The synergistic PHSRN sequence and the RGD motif are highlighted in yellow and blue, respectively.

Fig 5

RDCs and interdomain orientations of FIII9′10 variants. a, b, c, and d correspond to FIII9′10; e, f, g, and h correspond to FIII9′10-CC_red; and i, j, k, and l correspond to FIII9′10-CC_ox. The left panels a, e, i, and b, f, and j show for each variant the same sections of the spectra used to measure the apparent ¹J_NH couplings in isotropic solution (a, e, and i) and in a strained polyacrylamide gel (b, f, and j). The values of the ¹J_NH couplings are given in Hz, and for clarity, the resonances are labeled with amino acid names only in a. The middle panels c, g, and k show the correlation between the observed and calculated RDCs, and the right panels d, h, and l show the resulting interdomain orientation of the corresponding domain pairs.

Fig. 6

Backbone dynamics of FIII9′10 variants. Shown is a plot of [¹H]¹⁵N NOE for FIII9′10 (a), FIII9′10-CC_red (b), and FIII9′10-CC_ox (c). The filled bars denote the location of β-strands, and the dotted lines mark the sites of the mutated residues Ala¹³⁴⁰ and Val¹⁴⁴². The synergistic PHSRN sequence and the RGD motif are highlighted in yellow and blue, respectively.

Fig. 7

¹⁵N-T₁ and ¹⁵N-T₂ relaxation time constants for FIII9′10 (a), FIII9′10-CC_red (b), and FIII9′10-CC_ox (c). The closed circles indicate residues from the FIII9 domain, and open circles show residues from the FIII10 domain. The lines are theoretical T₁ and T₂ values calculated using the Lipari-Szabo model (44) with order parameters, S², of 1, 0.9, 0.8, 0.7, 0.6, and 0.5.