Conformational dynamics in loop swap mutants of homologous fibronectin type III domains

Abstract

Fibronectin type III (FN-III) domains are autonomously folded modules found in a variety of multidomain proteins. The 10th FN-III domain from fibronectin (fnFN10) and the 3rd FN-III domain from tenascin-C (tnFN3) have 27% sequence identity and the same overall fold; however, the CC' loop has a different pattern of backbone hydrogen bonds and the FG loop is longer in fnFN10 compared to tnFN3. To examine the influence of length, sequence, and context in determining dynamical properties of loops, CC' and FG loops were swapped between fnFN10 and tnFN3 to generate four mutant proteins and backbone conformational dynamics on ps-ns and mus-ms timescales were characterized by solution (15)N-NMR spin relaxation spectroscopy. The grafted loops do not strongly perturb the properties of the protein scaffold; however, specific effects of the mutations are observed for amino acids that are proximal in space to the sites of mutation. The amino acid sequence primarily dictates conformational dynamics when the wild-type and grafted loop have the same length, but both sequence and context contribute to conformational dynamics when the loop lengths differ. The results suggest that changes in conformational dynamics of mutant proteins must be considered in both theoretical studies and protein design efforts.

FIGURE 1

Fibronectin type 10 domain structure and sequence alignments. The structural representation is drawn using Molscript (48) from the PDB file 1FNA; five N-terminal residues not present in the crystal structure have been built onto the structure (17). The sequence alignment uses the fnFN10 residue numbering; thus, two-residue gaps appear before and after the FG loop RGD sequence in tnFN3. The β-strands are indicated by bars above the fnFN10 and tnFN3 sequences and by boldfaced letters in the sequence alignment. Conserved residues in the mutant proteins are shown by dots; gaps or deletions are shown by dashes.

FIGURE 2

Chemical shift perturbations. The backbone chemical shift perturbation for backbone ¹⁵N and ¹H^N nuclei, defined by in which Δδ_X is the difference in the chemical shift of nucleus X in the mutant, and wild-type proteins are shown as a function of amino acid sequence for (a) fnTNCC, (b) fnTNFG, (c) tnFNCC, and (d) tnFNFG proteins. Data for the mutated residues are shown in gray. The locations of the β-strands from A to G are indicated by the horizontal gray bars.

FIGURE 3

Backbone dynamics on ps-ns timescales. Values of backbone ¹⁵N S² for loop swap mutant proteins are compared to values for wild-type fnFN10 and tnFN3. (a) fnTNCC (blue circles and solid line) and (b) fnTNFG (red circles and solid line) are compared to wild-type fnFN10 (black circles and solid line) and wild-type tnFN3 (black triangles and dashed line). (c) tnFNCC (blue circles and solid line) and (d) tnFNFG (red circles and solid line) are compared to wild-type tnFN3 (black circles and solid line) and wild-type fnFN10 (black triangle and dashed line). Error bars similar to or smaller than the size of the plotted points are not shown. Expansions showing the site of mutation are shown: (e) fnTNCC residues 30–50, (f) fnTNFG residues 70–90, (g) tnFNCC residues 30–50, and (h) tnFNFG residues 70–90. Error bars are not shown for clarity.

FIGURE 4

Structural dependence of backbone dynamics on ps-ns timescales. The values of S² shown in Fig. 3 are mapped in pseudocolor onto the fnTNCC predicted structure, the fnFN10 structure, the fnTNFG predicted structure, the tnFNCC predicted structure, the tnFN3 structure, and the tnFNFG predicted structure. The location of the CC′ and FG loops are shown for the wild-type structures. The first five residues were not present in the PDB file 1FNA used to perform the data analysis for wild-type fnFN10.

FIGURE 5

Backbone dynamics on μs-ms timescales. Values of R_ex for loop swap mutant proteins are compared to values for wild-type fnFN10 and tnFN3. (a) fnTNCC (blue circles and solid line) and (b) fnTNFG (red circles and solid line) are compared to wild-type fnFN10 (black circles and solid line). (c) tnFNCC (blue circles and solid line) and (d) tnFNFG (red circles and solid line) are compared to wild-type tnFN3 (black circles and solid line). Error bars similar to or smaller than the size of the plotted points are not shown. Expansions showing the site of mutation are shown: (e) fnTNCC residues 30–50, (f) fnTNFG residues 70–90, (g) tnFNCC residues 30–50, and (h) tnFNFG residues 70–90. In (e and f) R_ex for wild-type tnFN3 is depicted using black triangles and dashed line. (g and h) R_ex for wild-type fnFN10 is depicted using black triangles and dashed line.