Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates

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 <i>N</i>-methyl Gly (sarcosine) in the P1′ subsite and <i>N</i>-isobutyl Gly (<i>N</i>Leu) 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

Conformational Dynamics of Matrix Metalloproteinase-1·Triple-Helical Peptide Complexes

Matrix metalloproteinase-1 (MMP-1) is a zinc-dependent protease that catalyzes hydrolysis of interstitial collagens. A previously reported X-ray crystallographic structure revealed specific interactions between a triple-helical peptide (THP) model of interstitial collagen and the hemopexin-like (HPX) and catalytic (CAT) domains of MMP-1. An NMR-based structure of MMP-1 in a complex with a different THP was also solved, where docking was used to model the MMP-1·THP interactions and develop a mechanism for the early stages of collagenolysis. To provide greater insight into and reveal specific details of the collagenolytic mechanism, molecular dynamics (MD) studies of the MMP-1·THP NMR-derived and X-ray crystallographic complexes were performed and compared. The “open/extended” conformation of the NMR-derived MMP-1·THP complex was found to lead to a catalytically productive complex. The X-ray crystallographic MMP-1·THP complex was initially in a “closed/collapsed” conformation, and did not yield a productive complex. The NMR-derived structure of the MMP-1·THP complex possessed many more atomistic interactions between MMP-1 and the THP compared with the X-ray crystallographic structure of the MMP-1·THP complex, and also had greater participation of MMP-1 in the local unwinding/destabilization of the THP. The atomistic interactions support the favorable energetics of the initial step of collagenolysis originating from the NMR-derived MMP-1·THP complex structure

Effects of δ/ω-PLTX-Pt1a (20 nM) on the voltage-dependent Na⁺ currents.

<p>Results are shown as Mean±SD. * p<0.01. **p<0.002. (Paired student's t-test).</p><p><b>a</b>. V_th is the threshold membrane potential at which the amplitude of inward Na⁺ current is larger than 50 pA which can be confidently distinguished from the noise level;</p><p><b>b</b>. V_h is the membrane potential of half-inactivation;</p><p><b>c</b>. I_p and I_ss are the amplitudes of peak and steady-state Na⁺ currents respectively;</p><p><b>d</b>. ♦is the decay rate of the inactivating Na⁺ current evoked by depolarizing pulse to −20 mV from the holding potential of −80 mV. The value was obtained by single exponential fitting of the current decay phase. In 2 of the 14 cells, currents were better fitted with a double-exponential function, of which ♦₁ was shown here.</p><p><b>e</b>. r is the fractional recovery from inactivation of Na⁺ currents at −10 mV, assessed by the double pulse paradigm with an interpulse interval of 500 ms.</p

Excitatory effect of δ/ω-PLTX-Pt1a at <i>Drosophila</i> neuromuscular junctions.

<p><b>A</b>. Control EJP recorded from the muscle of a third-instar larvae elicited by nerve stimulation. δ/ω-PLTX-Pt1a progressively prolonged stimulus evoked EJPs. Recordings were made 1 min (<b>B</b>), 3 min (<b>C</b>), and 5 min (<b>D</b>) after the application of 5 nM δ/ω-PLTX-Pt1a. <b>E</b>. Simultaneous intracellular recordings (top trace) from the larval muscle and extracellular recordings (bottom trace) from the motor nerve near its terminal region under control condition. <b>F</b>. In the presence of δ/ω-PLTX-Pt1a, a prolonged EJP is associated with a train of spikes originated at or near the nerve terminal. Representative figures from one of more than 20 independent experiments are shown.</p

Purification of δ/ω-PLTX-Pt1a.

<p><b>A</b>, Crude <i>Plectreurys</i> venom was fractionated on a HPLC G-50 column. Further purification was done with the fraction (▴) taken from the later part of the second complex peak. Insect-specific inhibitory toxins including PLTX II were previously purified from another G50 fraction (▾). <b>B</b>, The G-50 fraction was separated by RP-HPLC on a C18 column and eluted with a gradient of 30% to 60% ACN in 0.1% TFA, as shown by the dashed line. <b>C</b>, The predominantly active fraction (indicated by the arrow in <b>B</b>) was further purified on an analytic C18 column in a second solvent system containing 0.1% HFBA, which separates δ/ω-PLTX-Pt1a (arrow) from two inhibitory peaks (▴).</p

Structural characterization of δ/ω-PLTX-Pt1a.

<p><b>A</b>. Native δ/ω-PLTX-Pt1a was subjected to alkaline depalmitoylation with 1N NaOH (pH 10.0) for 60 min, and analyzed by RP-HPLC with a gradient of 0–60% ACN in 0.1% TFA. <b>B</b>. Fragments generated from trypsin digestion of reduced and pyridethylated δ/ω-PLTX-Pt1a were separated on a C18 RP-HPLC column. Amino acid sequences for these fragments are given above each peak, followed by the masses predicted from their sequences (italic fonts) and the masses measured by mass spectrometry. The asterisks (*) indicate fatty modification at the C-terminal fragment.</p

Effects of δ/ω-PLTX-Pt1a on voltage-dependent Na⁺ currents.

<p>Representative whole-cell Na⁺ currents recorded from a cultured <i>Drosophila</i> neuron, before (<b>A</b>) and after the application of 20 nM δ/ω-PLTX-Pt1a (<b>B</b>). Na²⁺ currents were evoked by a series of depolarizing voltage steps in 10 mV increments from a holding potential of −80 mV. (<b>C</b>) To compare the voltage-dependence of Na⁺ channel activation, peak Na⁺ currents before (open circles) and after δ/ω-PLTX-Pt1a (solid circles) were normalized to the maximum amplitude (I/Imax) and plotted against test potentials. (n = 7; mean ± S.E.). <b>D</b>. Representative Na⁺ current traces were normalized and aligned to show a slowing of channel inactivation by δ/ω-PLTX-Pt1a.</p

δ/ω-PLTX-Pt1a sequences.

<p><b>A</b>. Complete amino acid sequence of δ/ω-PLTX-Pt1a. Pal: O-palmitoyl moiety; amide: amidated C-terminal. <b>B</b>. cDNA coding sequence and amino acid sequence of δ/ω-PLTX-Pt1a. The mature toxin is indicated by bold fonts, and the amidation signal sequence at the C-terminus is underlined. The arrow denotes a putative signal cleavage site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064324#pone.0064324-Leisy1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064324#pone.0064324-Penaforte1" target="_blank">[27]</a>. <b>C</b>. Comparison of amino acid sequences of δ/ω-PLTX-Pt1a, ω-PLTX-Pt1a (PLTX II) and U₁-PLTX-Pt1a (Plt-VI). Plt-VI was reported by Quistad and Skinner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064324#pone.0064324-Quistad2" target="_blank">[7]</a>. Identical residues in all three peptides are shown in <i>shaded boxes</i>. Spacers (-) are inserted to show maximal homology. The asterisks (*) denote O-palmitoylated residues.</p

δ/ω-PLTX-Pt1a reduces stimulus-induced intracellular Ca²⁺ rise in cultured <i>Drosophila</i> neurons.

<p>Summary of average decreases in high K<b>⁺</b> induced intracellular Ca²⁺ rises produced by δ/ω-PLTX-Pt1a and ω-PLTX-Pt1a (PLTX II). 20 nM δ/ω-PLTX-Pt1a blocked a portion of stimulus-induced intracellular Ca²⁺ rises, while 5 nM ω-PLTX-Pt1a nearly blocked all the Ca²⁺ rises in cultured <i>Drosophila</i> embryonic neurons. Values are shown as mean ± S.E.</p

δ/ω-PLTX-Pt1a partially blocks neuronal voltage-dependent Ca²⁺ currents.

<p>Representative whole-cell Ca²⁺ currents recorded from a cultured <i>Drosophila</i> neuron, before (<b>A</b>) and after (<b>B</b>) the application of 100 nM δ/ω-PLTX-Pt1a. Ca²⁺ currents were evoked by a series of depolarizing voltage steps in 10 mV increments from a holding potential of −80 mV. For clarity, only three of the traces were shown. (<b>C</b>) and (<b>D</b>) show the I-V relations of peak and steady-state Ca²⁺ currents before (open symbols) and after addition of δ/ω-PLTX-Pt1a (solid symbols). Representative figures from one of five independent experiments are shown.</p