Interdomain flexibility in full-length matrix metalloproteinase-1 (MMP-1)

Abstract

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs.

FIGURE 1.

¹H-¹⁵N-TROSY-HSQC spectra of FL-MMP-1 (²H-¹³C-¹⁵N-labeled NNGH-inhibited, E219A mutant) (black), superimposed with CAT-MMP-1 (¹⁵N-labeled NNGH-inhibited, E219A mutant) (red) and with HPX-MMP-1 (¹⁵N-labeled) (green).

FIGURE 2.

Plot of mean shift differences experienced by FL MMP-1 with respect to the isolated CAT or HPX domains. avg. indicates average.

FIGURE 3.

Mapping of the residues at the interface between the CAT domain, the linker and the HPX domain. A, exploded space fill representation of the experimental three-dimensional x-ray structure of FL-MMP-1 (24) where the regions of contact between CAT and HPX domains are colored in green and the regions of contact of either of the two domains with the linker are colored in red. The arrangement of the two domains and the linker in the crystal structure is visible in the background. B, the same exploded space fill representation where the intensity of the colored residues, from yellow to dark-orange, correlates with the size of the mean shift difference experienced by FL-MMP-1 with respect to the isolated domains. The similarity of the areas experiencing shift changes due to the lack of the linker in B with the contact areas in red, but not with those in green in A, show that most of the shift perturbation arises from the contacts with the linker and not between the two domains.

FIGURE 4.

Comparison of NMR relaxation data for MMP-1 (data collected at 306 K) and MMP-12 (data collected at 298 K). The calculated (gray bars) and experimental (filled circles) backbone ¹⁵NH R₁ and R₂ values for the isolated CAT and HPX domains of MMP-1 are shown in A and C, and those of MMP-12 are shown in F and H. The ¹⁵NH R₁ and R₂ values for the full-length MMP-1 and MMP-12 are shown in B, D, G, and I, respectively. The experimental NOE values for the full-length proteins are shown in E and L for MMP-1 and MMP-12, respectively. In both proteins, the agreement between experimental and calculated R₁ values for the isolated domains is excellent, whereas for the full-length proteins, the experimental R₁ values are sizably larger (B and G) than the ones calculated for the rigid x-ray structures. R₂ values (D and I) are also in good agreement if a slight tendency of the HPX domain to aggregate is taken into account. The data comparisons are only consistent with interdomain flexibility in MMP-1, which is somewhat less extensive than in MMP-12.

FIGURE 5.

Experimental x-ray scattering from the NNGH-inhibited, FL-MMP-1 and scattering calculated from the following models: curve 1, experimental data with error bars; curves 2 and 3, computed scattering from the crystallographic models of FL-MMP-1 and FL-MMP-12, respectively; and curve 4, a typical fit by the selected ensemble of structures. The logarithm of the scattering intensity is plotted against the momentum transfer. Inset, the frequency of the models with the given R_g in the initial pool of structures with randomized interdomain linkers (FL-MMP-1 (blue) and FL-MMP-12 (magenta)) and in the selected ensembles (FL-MMP-1 (red) and FL-MMP-12 (green); the latter distribution is obtained by the averaging of several EOM runs. All histograms are normalized to the integral value of unity.