Structural Basis for Matrix Metalloproteinase 1-Catalyzed Collagenolysis

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme–substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782–785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray “closed” conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function

Data collection statistics for native and selenomethionine substituted AmyR.

<p>Values in parentheses are for the highest resolution shell.</p

Refinement statistics for native and selenomethionine substituted AmyR.

<p>Refinement statistics for native and selenomethionine substituted AmyR.</p

Unmodelled density at crystal contact between molecules.

<p>(A) Electron density and difference map of the unmodelled density at the crystal contact. The main crystal contact forming density is central, while the two other chains are seen below. (B) An alternative view of the two chains.</p

Structural alignment of AmyR monomer with other YbjN and T3C proteins.

<p>The two views are rotated horizontally 90°. The alignment includes the PDB entries 1JYO:D (<i>Salmonella enterica</i> SicP, ice blue), 1S28:A (<i>Pseudomonas savastanoi</i> AvrPphF ORF1, gold), 1TTW:A (<i>Yersinia pestis</i> SycH, coral), 1XKP:B (<i>Yersinia pestis</i> SycN, grey), 2PLG:B (<i>Synechococcus elongates</i> T110839, pink), 3EPU:A (<i>Salmonella enterica</i> STM2138, sea green), 3KXY:J (<i>Escherichia coli</i> ExsC, brown), and 4H5B:A (<i>Deinococcus radiodurans</i> DR_1245, lilac), and 5FR7:A (<i>Erwinia amylovora</i> AmyR, yellow).</p

Rotated views of the <i>E</i>. <i>amylovora</i> AmyR dimer.

<p>(A) Ribbon diagram, coloured by secondary structure. (B) Surface topography, coloured by surface charge at pH 7.4, red = negative, blue = positive.</p

Ribbon representation of the overall structure of the AmyR monomer.

<p>The AmyR monomer is coloured by secondary structure. Secondary structure elements are labelled with residue numbers. The right panel is rotated 90°. β-strand 1 interacts with the equivalent residues in the dimer, shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176049#pone.0176049.g002" target="_blank">Fig 2</a>.</p

The water molecules between the dimer interface.

<p>Cross-section of the hydrophilic pocket at the dimer interface, showing the A and B chains coloured in gold and silver, with the highly ordered water molecules contained within.</p

The crystal structure of <i>Erwinia amylovora</i> AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions

<div><p>AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species <i>Erwinia amylovora</i>, and is a functionally conserved ortholog of YbjN from <i>Escherichia coli</i>. The crystal structure of <i>E</i>. <i>amylovora</i> AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.</p></div

Location of conserved regions and residues of YbjN proteins superimposed on <i>E</i>. <i>amylovora</i> AmyR structure.

<p>(A) Sphere representation of AmyR displaying the two chains of the dimer in grey and green, with the atoms of conserved sidechains shown in red Van der Waals spheres. The main chains are partially transparent to show that the conserved residue side chains are mostly internalised or at the dimerization interface. (B) The same view as a ribbon diagram, with the YbjN protein conserved regions in red.</p