Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

Abstract

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.

Figure 1.

NMR ensemble of human MIA shown as a spline with variable radius, representing the RMS of the deviation from the mean structure (Stoll et al. 2001). The N and C termini are indicated by N and by C, respectively. This figure was generated with MOLMOL and POV-Ray (http://www.povray.org) (Koradi et al. 1996).

Figure 2.

Steady-state heteronuclear ¹⁵N{¹H}-NOE for the backbone amides of human MIA. Residues for which no results are shown correspond either to prolines or to residues where relaxation data could not be extracted. For calculation of experimental errors, refer to materials and methods. The arrows indicate β-strands as described previously (Stoll et al. 2001).

Figure 3.

Plots of ¹⁵N longitudinal relaxation rates R₁ (A) and transverse relaxation rates R₂ (B) of MIA as a function of the residue number at 300 K. For details, refer to text.

Figure 4.

Cross correlation rates η for human MIA. For calculation of experimental errors, refer to Materials and Methods.

Figure 5.

Microscopic parameters of motion of S² (A) and R_ex (B) for MIA. For details, refer to text.

Figure 6.

Ribbon of a NMR structure of human MIA representative for the ensemble. The N and C termini are indicated by N and by C, respectively. The color shading is based on the values of the order parameter S² (A) and for the exchange rates R_ex (B). This figure was generated with Insight II (www.accelrys.com).

Figure 7.

Inhibition of attachment of melanoma cells by different mutants of human MIA. The following point mutations have been used in the inhibition bioassay: (1) Wild-type MIA, (2) V49I/R56S, (3) L53Q, (4) G62R, (5) E17V/D72A, (6) E17G/A74P, (7) T90P, (8) dC-term 82, (9) dC-term 80, (10) dC-term 74, (11) dC-term 67. For details, refer to Materials and Methods.

Figure 8.

Sequence alignment of human MIA (1HJD), bovine MIA, mouse MIA, rat MIA, human MIA-LIKE (MIAL), mouse MIAL, human fibrocyte-derived protein (FDP), and mouse FDP including their signal peptides. The numbering of amino acids follows the sequence of human MIA as previously published (Stoll et al. 2001). Conserved residues are boxed. The color code reflects the physical and chemical properties of the amino acids and was taken from the Jalview program (http://www.ebi.ac.uk). Aromatic and hydrophilic residues are colored in light blue and blue, positively charged residues are red, negatively charged residues are magenta, hydrophilic residues are green, glycines are orange, prolines are colored in yellow, and cysteines are colored in light red. For details, refer to the discussion. This figure was generated with the Swissprot software package (http://www.expasy.ch) and Jalview (http://www.ebi.ac.uk) (Appel et al. 1994).