Apparent Tradeoff of Higher Activity in MMP-12 for Enhanced Stability and Flexibility in MMP-3

Liang, Xiaogan; Arunima, A.; Zhao, Yunxin; Bhaskaran, R.; Shende, Anuradha; Byrne, Todd S.; Fleeks, Jeremy; Palmier, Mark O.; Doren, Steven R. Van

doi:10.1016/j.bpj.2010.04.002

Cited by 14 publications

(22 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Use of E219A-inactivated MMP-12 prevented digestion of Eln-20. The extent of this structural perturbation is localized to residue 219 in the active site (49,(71)(72).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

Palmier

Fulcher

Bhaskaran

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that ␣-elastin species cover the primed subsites, a strip across the ␤-sheet from ␤-strand IV to the II-III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the "primed" side in the III-IV, V-B, and S1 specificity loops. Two map to the "unprimed" side in the IV-V and B-C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.Although protein-protein interactions are universally important, mechanistic understanding of their specificity is often poor (1). An impediment to detailed understanding of proteolytic attack of proteins is the transience and potential heterogeneity of the interactions, which interfere in capturing the structure of a substrate complex by crystallography or other methods. These complications affect characterization of matrix metalloproteinase-12 (MMP-12), 3 the metalloelastase secreted by human macrophages at sites of inflammation. To investigate how MMP-12 achieves specificity for protein fibrils from lungs and arteries, we developed an approach designated BINDSIght, for its combination of bioinformatics and NMR discovery of specificity of interactions.In lungs, arteries, skin, and basement membranes, elastin provides elastic recoil, is heavily cross-linked, and is difficult to digest. Collagens are ubiquitous and comprise ϳ25% of the protein mass of the body. Damage to fibrils of the extracellular matrix by proteases such as MMP-12 contributes to the inflammation and chronic disease states of chronic obstructive pulmonary disease (2-4), atherosclerosis (5-6), abdominal aortic aneurysm (7), multiple sclerosis (8), ulcerative colitis (9), asthma (4), and rheumatoid arthritis (10). The progression of chronic obstructive pulmonary disease/emphysema and (abdominal aortic aneurysm) in smokers depends in large part on MMP-12 expression (11) and its degradation of elastin (7, 12). ...

show abstract

“…Use of E219A-inactivated MMP-12 prevented digestion of Eln-20. The extent of this structural perturbation is localized to residue 219 in the active site (49,(71)(72).…”

Section: Resultsmentioning

confidence: 99%

“…5, a and c) to account for the high elastase activity of MMP-12 is that its advantage over MMP-3 is not in K m , but rather 13.5-fold in k cat ( Table 1). The lower k cat of MMP-3 could be related to its greater extent of millisecond motions surrounding the active site (72).…”

Section: Of Ref 33)mentioning

confidence: 99%

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

Palmier

Fulcher

Bhaskaran

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…We set out to discover whether the well-characterized MMP-12 22, 23, 24, 25, 26 binds directly to lipid bilayers and plasma membranes of living cells. Our results take on unexpected relevance to the recent question of how active MMPs enter and move through cells 2, 27, 28 .…”

mentioning

confidence: 99%

Ambidextrous binding of cell and membrane bilayers by soluble matrix metalloproteinase-12

et al. 2014

Self Cite

View full text Add to dashboard Cite

Matrix metalloproteinases (MMPs) regulate tissue remodeling, inflammation, and disease progression. Some soluble MMPs are inexplicably active near cell surfaces. Here, we demonstrate binding of MMP-12 directly to bilayers and cellular membranes using paramagnetic NMR and fluorescence. Opposing sides of the catalytic domain engage spin-labeled membrane mimics. Loops project from the β-sheet interface to contact the phospholipid bilayer with basic and hydrophobic residues. The distal membrane interface comprises loops on the other side of the catalytic cleft. Both interfaces mediate MMP-12 association with vesicles and cell membranes. MMP-12 binds plasma membranes and is internalized to hydrophobic perinuclear features, the nuclear membrane, and inside the nucleus within minutes. While binding of TIMP-2 to MMP-12 hinders membrane interactions beside the active site, TIMP-2-inhibited MMP-12 binds vesicles and cells, suggesting compensatory rotation of its membrane approaches. MMP-12 association with diverse cell membranes may target its activities to modulate innate immune responses and inflammation.

show abstract

“…9A). 15 N relaxation experiments have shown them to be flexible (59). Intriguingly, R 1 measurements (60) showed that both these areas, which together form the upper and lower rims of the narrowest part of the active site (Fig.…”

Section: Resultsmentioning

confidence: 99%

Path to Collagenolysis

Prior

Byrne

Tokmina‐Roszyk

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ϳ1,080 Å 2 of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile GϳVV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling.Collagens comprise around 30% of the protein mass of the body (1) and resist general proteolysis. Matrix metalloproteinases (MMPs) 2 degrade collagen during cancer cell migration (2), angiogenesis (3, 4), destabilization of atherosclerotic plaques (5), and arthritis (6) and after myocardial infarction (7). For instance, plaques contain a collagen-rich cap and are sensitive to MMP-12 secreted by macrophages that makes them more vulnerable to rupture, precipitating myocardial infarction, or stroke (8).Collagen fibrils are bundles of staggered triple helices. Each triple helix comprises three extended strands twisted into a right-handed superhelix with hydrogen bonding between chains (9). Each collagen chain is a long series of GXY triplets in which the glycine is in the interior, X is often proline, and Y is often a stabilizing 4-hydroxyproline (Hyp). Because each chain is offset by one residue, the chains have been named leading, middle, and trailing (10). Lack of Pro and Hyp in four GXY triplets on the C-terminal side of the MMP scissile bond in interstitial collagens (types I, II, and III) was proposed to loosen the triple helix (1); this was later observed as 10-fold symmetry (11).Protease substrates are labeled by increasing distance from the scissile peptide bond with a prime indicating the C-terminal side: P 4 -P 3 -P-P 1 ϳP 1 Ј-P 2 Ј-P 3 Ј-P 4 Ј where ϳ denotes the scissile peptide bond. We refer to the residues on the N-terminal side of the scissile bond as "unprimed" and those on the C-terminal side as "primed." The...

show abstract

Apparent Tradeoff of Higher Activity in MMP-12 for Enhanced Stability and Flexibility in MMP-3

Cited by 14 publications

References 55 publications

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

Ambidextrous binding of cell and membrane bilayers by soluble matrix metalloproteinase-12

Path to Collagenolysis

Contact Info

Product

Resources

About