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Cooperative hydrophobic interactions between collagen and collagenase underlie collagen unwinding

Szymon W. Manka¹, Robert Visse¹, Dominique Bihan², Richard W. Farndale², Gillian Murphy³, Jan J. Enghild⁴ and Hideaki Nagase¹
¹Kennedy Institute of Rheumatology, Faculty of Medicine, Imperial College London, W6 8LH, UK, ²Department of Biochemistry, University of Cambridge, CB2 1QN, UK, ³Department of Oncology, University of Cambridge, Cancer Research UK Cambridge Institute, Li Ka Shing Centre, CB2 0RE, UK, ⁴Laboratory for Proteome Analysis and Protein Characterisation, Department of Molecular Biology, University of Aarhus, DK-8000, Denmark

Introduction
Collagen turnover is key in vertebrate tissue homeostasis. Unwanted or insufficient collagenolysis interrelates with diseases such as arthritis, fibrosis, atherosclerosis or cancer. Due to the triple-helical fold interstitial collagens are resistant to most proteases. However, selected members of the matrix metalloproteinase family (MMP-1, -2, -8, -13 and -14) cleave fibril-forming collagen I, II and III molecules at one specific site, approximately 3/4 away from the N-terminus. Collagenases (MMP-1, -8, and -13) comprise two domains, the N-terminal catalytic domain (Cat) and the C-terminal 4bladed β-propeller hemopexin domain (Hpx), connected via a flexible linker (hinge) peptide. Both domains are required for triple-helical collagen cleavage. Prior to peptide bond hydrolysis collagen triple-helix needs to be locally unwound, as the active site cleft of collagenase is too narrow to accomodate the whole collagen molecule, and the scissile bond is masked in a triple-helical arrangement of the substrate. However, the detailed mechanism of collagenolysis by MMPs remaines elusive.
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Leu at the P1’ and P10’ subsites in interstitial collagens are pivotal for collagenase binding
A 450
0.3

*proMMP-1(E200A) *MMP-1(E200A) *Hpx *Cat(E200A)

Toolkit II

0.2

0.1

We screened the THP library encompassing the entire collagen II sequence using MMP-1(E200A) and its individual domains. The only THP that bound to MMP-1(E200A) with high affinity was Col II-44, which starts at the collagenase cleavage site sequence G#LAGQR (# is the bond cleaved by MMP-1). We then tested a series of Col II-44 derivatives and found that THPs missing the first (P1’) or the second (P10‘) Leu of the peptide, showed profound reduction in MMP-1(E200A) binding (marked with *). Alanine scanning mutagenesis of DB133 peptide revealed that the two Leus at the P1’ and P10’ position are key for MMP-1(E200A) binding. An aligment of this region in all α chains of collagen I, II and III from different species (not shown) indicated that the two Leus at P1’ and P10’ sites are conserved and that the consensus sequence is G#(L/I)(A/L)-GXY-GXY-GL(O/A) where the triplets containing conserved Leu/Ile are separated by two GXY triplets (X,Y - any amino acid).

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 GPP BSA

In this study we addressed the stuctural and mechanistic basis of collagenolysis. We aimed to find out how collagenase interacts with collagen: what are the contact sites between the enzyme and the substrate and how is the cleavage site in collagen recognised.

* Col.II-43 GPC(GPP)5-GEOGDDGPSGAEGPOGPQGLAGQRGIV-(GPP)5GPC GPC(GPP)5-GLAGQRGIVGLOGQRGERGFOGLOGPS-(GPP)5GPC Col.II-44 GPC(GPP)5-GFOGLOGPSGEOGKQGAOGASGDRGPO-(GPP)5GPC Col.II-45
*MMP-1(E200A) binding level (A450) DB122 DB108 DB123 DB124 NR393 NR394 DB125 DB126 NR373 DB133 DB147 DB148 DB135 DB151 DB138 DB139 DB140 DB141BIS DB142 DB143 DB144 DB145 DB150 GPC(GPP)5-GLAGQRGIVGLOGQRGER-(GPP)5GPC GPC(GPP)5-GFOGLOGPS-(GPP)5GPC * GPC(GPP)5-GQRGIVGLOGQRGER-(GPP)5GPC * GPC(GPP)5-GIVGLOGQRGER-(GPP)5GPC * GPC(GPP)5-GLAGQRGIVGLOGQR-(GPP)5GPC GPC(GPP)5-GLAGQRGIVGLO-(GPP)5GPC GPC(GPP)5-GQRGIVGLO-(GPP)5GPC * GPC(GPP)5-GIVGLOGQR-(GPP)5GPC * GPC(GPP)5-GLOGQRGERGFO-(GPP)5GPC * GPC(GPP)5-GPQGLAGQRGIVGLOGQRGER-(GPP)5GPC GPC(GPP)5-GAQGLAGQRGIVGLOGQRGER-(GPP)5GPC GPC(GPP)5-GPAGLAGQRGIVGLOGQRGER-(GPP)5GPC * GPC(GPP)5-GPQGAAGQRGIVGLOGQRGER-(GPP)5GPC GPC(GPP)5-GPQGLAGARGIVGLOGQRGER-(GPP)5GPC GPC(GPP)5-GPQGLAGQAGIVGLOGQRGER-(GPP)5GPC GPC(GPP)5-GPQGLAGQRGAVGLOGQRGER-(GPP)5GPC GPC(GPP)5-GPQGLAGQRGIAGLOGQRGER-(GPP)5GPC * GPC(GPP)5-GPQGLAGQRGIVGAOGQRGER-(GPP)5GPC GPC(GPP)5-GPQGLAGQRGIVGLAGQRGER-(GPP)5GPC GPC(GPP)5-GPQGLAGQRGIVGLOGARGER-(GPP)5GPC GPC(GPP)5-GPQGLAGQRGIVGLOGQAGER-(GPP)5GPC GPC(GPP)5-GPQGLAGQRGIVGLOGQRGAR-(GPP)5GPC GPC(GPP)5-GPQGLAGQRGIVGLOGQRGEA-(GPP)5GPC
P4 P1’ P10’ P17’

Collagenase cleavage site

MMP-1 binds to collagen I through the cooperative action of the Cat and the Hpx domains
We first examined the abilities of MMP-1(E200A) and its individual domains to bind to immobilised native collagen I at 20 °C. MMP-1(E200A) is an active site mutant that binds and unwinds collagen, but cannot cleave peptide bonds. Full-length MMP-1(E200A) bound to collagen I in a saturable manner, but the binding of the Hpx domain was only minimal. and the Cat(E200A) domain showed no stable binding.

++ ++ ++ ++ ++ ++ ++ +++ ++ ++ ++ +++ + +++ +

Collagenase recognises a motif which in the triple-helical structural arrangement forms two hydrophobic 3-pronged rings in a 9-residue distance.

Both domains of collagenase contain low-affinity collagen binding subsites which express high affinity to collagen only if they act in concert when the domains are physically connected.

Temperature-dependent MMP-1(E200A) binding to collagen I and involvement of the Cat domain
The binding of MMP-1(E200A) to collagen was increased in a temperature-dependent manner, but at 40°C (gelatin) it is severely reduced. In the presence of GM6001, an active site inhibitor, the temperatureenhanced collagen binding was abolished.

Predicting the mode of collagenase-collagen interaction
Cat Cat

DB133 AlaScan

II-44 derivatives

P1’ cluster

Y191F F188A F301Y L295S L338A/H339A R272A I271A/R272A F289A/Y290A/P291A D317A E365A

Considering the H/DXMS data (wireframed) and the importance of two Leus at the P1´ and P10´ positions, we have searched sites in MMP-1 they may interact with. A THP model consisting of two α1(I) and one α2(I) chains was docked onto the 3D structure of MMP-1 placing P1’ Leu close to the S1’ pocket and the P10’ Leu in a hydrophobic patch of the enzyme in the area identified by H/DXMS. Potential hydrophobic residues and other possible sites which may interact with collagen I including the P10’ Leu were subjected to mutation and their collagenolytic activities were examined. The most severe reduction of collagenase activity was observed with the single mutation of F310Y and the double mutation of I271A/R272A (Lauer-Fields et al., J Biol Chem. 2009 Sep 4;284(36):24017-24) which exhibited 9.8 % and 3.6 % of the collagenolytic activity, respectively.

P10’ cluster

Hpx

Hpx
Activity (% WT) 100 74 115 3.6 36 76 41 9.8 122 114 144 ± ± ± ± ± ± ± ± ± ± ± 8 4.4 10.4 0.5 2.4 10.1 0.3 0.2 7.1 10.6 14.3

Thermally loosened triple-helices are favoured for MMP-1(E200A) binding up to a critical temperature above which the disorder in the helix is excessive. Temperature-enhanced binding involves the active site of the enzyme and may represent the complex with unwound collagen.

MMP-1 WT F188A Y191F I271A/R272A R272A F289A/Y290A/P291A L295S F301Y D317A L338A/H339A E365A

Rate (U*/ g enzyme) 13.9 10.2 16.0 0.5 5.0 10.5 5.6 1.4 17.0 15.8 20.0 ± ± ± ± ± ± ± ± ± ± ± 0.98 1.11 0.61 0.70 0.33 1.40 1.46 0.03 2.00 1.45 0.03

Mapping of the collagen-binding sites in MMP-1 by H/DXMS
Protection by collagen
81 92 102

No change
112 122

F VLTEGNPRWE QTHLTYRIEN YTPDLPRADV DHAIEKAFQL WSNVTPLTFT
132

KVSEGQADIM ISFVRGDHRD NSPFDGPGGN LAHAFQPGPG IGGDAHFDED
182 192 202

1

162

172

ERWTNNFREY NLHRVAAHAL GHSLGLSHST DIGALMYPSY TFSGDVQLAQ DDIDGIQAIY GRSQNPVQPI GPQTPKACDS KLTFDAITTI RGEVMFFKDR
242 232 252 262

2

222

3

FYMRTNPFYP EVELNFISVF WPQLPNGLEA AYEFADRDEV RFFKGNKYWA VQGQNVLHGY PKDIYSSFGF PRTVKHIDAA LSEENTGKTY FFVANKYWRY DEYKRSMDPG YPKMIAHDFP GIGHKVDAVF MKDGFFYFFH GTRQYKFDPK
382 402 422 392 412

4

Hydrogen/deuterium exchange mass spectrometry (H/DXMS) measures exchange of peptide amide hydrogen with the one from the solvent. In D₂O, solvent-exposed protons exchange to twice as heavy deuterons, and this is determined by MS after protein fragmentation, as is the peptide ID (MS/MS). The D buidup is reduced at the sites protected by ligand binding. We measured D incorporation in a free and collagen-bound MMP-1(E200A) over 5 h. The figure summarises the local H/D exchange behavior in the enzyme upon interaction with collagen. All peptides are indicated with the bars underneath the sequence of MMP-1(E200A). Five distinct regions (1-5) were protected by collagen, two in the Cat domain, below and above the active site cleft, and three in the Hpx domain blades 1 and 2. The minimal binding sites are demarcated in the primery and 3D Van der Waals surface structure and the hypothetical collagen binding axis is shown with a dashed line for two different orientations of MMP-1(E200A). Sites in the Hpx domain showed stronger, more stable protection by collagen.

U - g collagen cleaved per min

F301 and I271-R272 residues in MMP-1 are essential for the interaction with collagen.

Conclusions
o Two domains of collagenase act together in collagen recognition o Collagenase prefers somewhat slacker (but not melting) triple-helix as a substrate to a compactly folded one o Two 3-pronged hydrophobic rings at positions P1’ and P10’ are key for collagenase binding to collagen o Collagen binding regions are located around the active site cleft and in the Hpx domain blades 1 and 2. F301 and I271/R272 proved to play important role in the interaction with collagen o Predicted mode of collagen binding by collagenase confirmed by X-ray diffraction studies (Frederico Carafoli & Erhard Hohenester)

302

312

322

5

352

362

372

TKRILTLQKA NSWFNCRKN
Cat
1

432

442

Cat

Zn2+
2 4 5 3

Discussion

We thank Noriko Ito for preparing MMP-1 mutant clones

Hpx

Hpx
Tilted orientation

Standard orientation

Collagen binding sites were reside in both the Cat and the Hpx domains of collagenase, and Hpx domain seems to interact with collagen more stably.

Hydrophobic interaction may be the driving force of the collagen unwinding as it may break the water shell surrounding collagen and in turn perturb interchain hydrogen bond ladders. The catalytic domain seems to interact with collagen dynamically, which can be facilitated by the hinge-bending motion between the domains. Thermal plasticity in collagen is probably a prerequisite for the high-affinity interaction with collagenase which may lead to collagen unwinding. The two 3-pronged hydrophobic ring motif itself is probably not sufficient for the productive interaction with collagenase. It needs to be situated in the specific sequence context to allow the productive fit to the enzyme. This would explain why fibrillar collagen molecules are cleaved at only one site, which is the imino-poor region of relatively relaxed helix. Thermal structural fluctuations coincide there with the hydrophobic ring motif. We propose that the collagenolysis is a result of the cross-talk between collagenase and collagen through dynamic structural interaction.