Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Womens Centre, Level 3, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
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Abstract |
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Keywords: cell adhesion/conformational stability/fibronectin/FIII domain/proline substitution
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Introduction |
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The type III modules of fibronectin all share a common framework (Huber et al., 1994; Potts and Campbell, 1994
). The crystal structure of human FIII7-10 reveals that the four FIII modules assume an extended rod-like structure with the RGD and synergy sites 34 Å apart (Leahy et al., 1996
). The solution structure for human FIII10 suggests that the conformation of the RGD site, which resides on a loop between the ß-strands F and G, is largely disordered and mobile (Main et al., 1992
; Dickinson et al., 1994
). Most of the residues of the PHSRN synergy site in FIII9 also reside on a loop (between the ß-strands C' and E) on the same surface of the molecule as the RGD site. Both loops protrude some distance away from the molecule. The calculated tilt and rotation angles between FIII9 and FIII10 are small compared with those of the FIII7-8 and FIII8-9 pairs (Leahy et al., 1996
). The buried interdomain surface area for human FIII910 (333 Å2) is also lower than for the other domain pair interfaces, suggesting a certain degree of mobility between the two domains. Indeed, the solution structure of the mouse recombinant FIII910 indicates a high degree of intermodule flexibility (Copié et al., 1998
).
Although the importance of the precise spatial relationship between the RGD loop and the FIII9 synergy site is not yet fully established, it is clearly critical for biological activity (Grant et al., 1997). Copié et al. have suggested that FIII910 acts as a flexible scaffold upon which the RGD and synergy sites exist in an ensemble of biologically active conformations (Copié et al., 1998
). However, the conformations of the RGD and synergy loops of human FIII910 in solution remain poorly defined.
A previous report (Copié et al., 1998) demonstrates that mouse FIII910 has increased conformational stability compared to the human module pair (Spitzfaden et al., 1997
) and is sufficiently stable for resolution of the solution structure by NMR, even though the sequence identity between mouse and human FIII9 is high (83%). The series of mutant human FIII910 proteins we describe here contain amino acid substitutions in FIII9 that were introduced according to differences between the mouse and human FIII9 amino acid sequences. The mutations were chosen with the aim of defining specific residues that can confer thermodynamic stability on FIII9 in the FIII910 pair and that have a key function in folding of the domain pair. Alignment of primary sequences highlighted five non-conservative and 10 semi-conservative or conservative amino acid mismatches that exist between the mouse and human FIII9 modules (Figure 1a
). Of these, three non-conservative amino acid substitutions, Ser for Phe1335, Ile for Arg1358 and Pro for Leu1408, were introduced either alone or in combination into the human FIII9 module (Figure 1b
) to produce seven mutants: L1408P, R1358I, F1335S, L1408P + R1358I, L1408P + F1335S, R1358I + F1335S and L1408P + R1358I + F1335S. Mutations were made following the Quickchange protocol (Stratagene). Residues Phe1366Ser1367 were not substituted with SerVal from mouse because these mutations were predicted to represent a minimal change. The semi-conservative and conservative amino acid differences between the mouse and human sequences were similarly ignored.
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Substitution of Phe1335 with Ser is detrimental to protein expression in Escherichia coli |
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Substitution of Leu1408 with Pro confers stability on FIII9 |
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Figure 3 shows the two-step equilibrium denaturation curves for wild-type human FIII910 and mutants L1408P, R1358I and L1408P + R1358I. The results are in good agreement with previous studies which have demonstrated that the initial step represents the unfolding of FIII9 and the second step the unfolding of FIII10 (Spitzfaden et al., 1997
; Altroff et al., 2001
). A large difference in thermodynamic stability was observed between the native FIII9 and FIII10 modules (
GH2O being 4.9 and 12.6 kcal/mol, respectively) (Table I
). None of the mutations effectively altered the stability of FIII10: the differences in the values of
GH2O and m for the FIII10 module can be accounted for by the error associated with the linear extrapolation of
G back to zero concentration of GdnHCl.
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Substitution of Leu1408 with Pro enhances the cell adhesive activity of FIII910 |
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We have reported the use of a rational approach to engineer human FIII910 mutants with minimal amino acid substitutions that confer increased conformational stability on FIII9. The main findings are that a single substitution of Leu1408 with Pro in human FIII910 increases both protein expression and conformational stability of the FIII9 domain and enhances the biological activity of FIII910. Our data indicate that Pro at this position has a key function in protein folding.
The solution properties of the L1408P mutant were greatly improved over those of wild-type FIII910. The Pro1408 residue is predicted to lie at the boundary between ß-strand G and the FG loop (Leahy et al., 1996). Sequence alignment of the FIII7, FIII8, FIII9 and FIII10 modules shows that a Pro residue is commonly found at the beginning or end of a predicted ß-strand and could thus have a role in the folding of the common type III fibronectin module.
Interestingly, three ProPro pairs exist in the mouse FIII910 pair, while none are present in the human FIII910 pair. Such ProPro pairs would be expected to restrict severely the local flexibility of the protein backbone because of the narrow range of dihedral angles allowed by the Pro residue. However, refolding of the human FIII10 module, which is rich in Pro residues, proceeds very rapidly (Plaxco et al., 1996), and this module has good solution and stability properties (Spitzfaden et al., 1997
). The presence of ProPro pairs may therefore contribute to the stability of mouse FIII910 and also explain why the L1408P substitution, which creates the Pro1407Pro1408 pair, confers enhanced solubility and stability on human FIII910. In addition, the presence of the ProPro pair in the synergy loop of mouse FIII9 may result in reduced flexibility compared with the human counterpart, although the solution structure of the latter has yet to be determined.
In correlation with improved structural stability, the biological activity of the L1408P mutant was likewise increased in comparison with wild-type human FIII910. This was not expected to result from a direct interaction between the introduced Pro residue and the FIII9 synergy site, since they lie on the opposite sides of the module. It should also be noted that neither the L1408P nor the R1358I mutation was expected to alter the FIII9/FIII10 interface and thereby the interdomain mobility, which has been shown to affect the domain pairs biological function (Grant et al., 1997). This study therefore provides an example of how single residue substitution distant from the binding site appears to alter biological activity via a long-range conformational change.
A modest increase in conformational stability of FIII9 was detected on substitution of Ile for Arg1358. The aliphatic side chain of Ile would not be expected to occupy the same position as the guanidium group of Arg, which, in the crystal structure, forms part of a cluster of four Arg and two Glu residues at the module surface. Despite this, the packing of the seven-ß-strand module does not appear to be significantly altered, because the R1358I mutant exhibits the same extent of unfolding (m) as the wild-type. The increase in stability could therefore be attributed to more extensive hydrophobic interactions within the Ile1358Ile1359 pair, assuming that any alteration to the electrostatic interactions within the cluster of Arg/Glu residues does not affect module packing or solvation.
In conclusion, we have designed a mutant human FIII910 domain pair containing a single amino acid substitution that has increased conformational stability of the FIII9 module. The enhanced solubility, stability and function of L1408P suggests that the proline in this position affects the global conformational stability of FIII9, including the synergy site. The L1480P substitution in the human FIII910 pair provides a further tool for the dissection of the structural properties that confer functional activity on FIII910.
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Notes |
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2 To whom correspondence should be addressed. E-mail: hmardon{at}molbiol.ox.ac.uk
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Acknowledgments |
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References |
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Received March 19, 2002; revised August 29, 2002; accepted September 4, 2002.