Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates

Abstract

Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

Figure 1

CD properties of triple-helical peptides and peptomers. (A) Thermal transition curves of triple-helical peptides and peptomers. Readings were taken at λ = 225 nm and normalized to fraction folded. (B) Melting point evaluation of triple-helical peptides and peptomers based on thermal transition curves.

Figure 2

Screening of the susceptibility of triple-helical peptides and peptomers toward MMP hydrolysis at 37 °C after 24 h as measured by RP-HPLC analysis.

Figure 3

Initial velocities (V_i) for MMP hydrolysis of (A) pfTHP-1 and (B) Ac-pfTHP-1 at 30 °C. Kinetic parameters were then determined for MMP-13 (table).

Figure 4

(A) Molecular modeling of MS-8 interacting with MMP-1. The P7′ residue (middle strand) is sterically hindered during interaction with the S7′ pocket (THP, green; CAT domain, magenta; HPX domain, yellow; HPX residues interacting with the THP, orange; P1′ residue, red; P7′ residue, cyan). (B) Sequence alignment of the α1 helix region of the HPX domain containing the S7′ pocket. The possible S7′ residue causing hindrance is colored blue and numbered in blue.

Figure 5

rmsd calculated for Cα atoms in (A) THP and (B) MS-8 during a 20 ns simulation time. Numbers correspond to snapshot structures depicted in Figure 6.

Figure 6

Structure snapshots of (A) THP and (B) MS-8 with high and low Cα rmsd values identified in MD simulations, numbered accordingly from a trajectory (panels A and B of Figure 5, respectively).

Figure 7

rmsf calculated for Cα atoms in (A) THP and (B) MS-8 during a 20 ns simulation time. The average fragment rmsf (dashed line) was calculated for residues 10–30.

Figure 8

Analysis of the MD simulation of the MMP-13·MS-8 complex. Panel A shows the relative HPX domain movement conjugated with simultaneous destabilization and/or unwinding of MS-8. Panel B shows the interaction of the P7′ NLeu782 residue with the S7′ subsite containing hydrophobic residues Phe326 and Tyr360. A video showing domain movement (morphing) between the beginning and end frames of a 5 ns trajectory is available as Supporting Information.

Figure 9

Comparison of MS-2, MS-7, MS-8, Ac-pfTHP-1, and THP sequences. Red is for the peptoid residue, magenta for the fluorophore/ quencher attached through the ε-amino group of Lys, and green for terminal fragments.