Domain Functions

The C-terminal domains aid unwinding

Calcium dependent recruitment domains at the C terminus (PKD,CBD-α,CBD-β) recruit and partly swell insoluble collagen without unwinding its triple helix structure. This helps to accelerate hydrolysis of insoluble collagen substrate at low component concentrations.

Figure.1 Collagen binding domain. The surface is cyan, with purple texturing on the portion that interacts with the collagen triple-helix, shown as yellow tubes. Key residues are labelled.

Collagen binding domains (Figure 1.) (in mammalian collagenases these domains are called hemopexin-like domains) are crucial for collagen binding, unwinding of the triple helix and having a good catalytic turnover rate.

TLP domains catalyse hydrolysis

Thermolysin is a metalloproteinase enzyme (an enzyme who’s catalytic mechanism involves a metal). Collagenase G has a TLP-like fold, that contains an HEXXH motif (that binds zinc) between residues 523 and 527. This region specifically catalyses the hydrolysis of peptide bonds containing hydrophobic residues.


There is an observed approximate 3A contraction of the two TLP half domains upon zinc loading and inhibitor binding. This tells us that correct positioning of the zinc atom occurs due to additional co-ordination by the substrate.


Glycine rich hinge region


A 40Å distance between these two domains is required to fit a collagen molecule (which remarkably has a diameter of around 40Å) also known as the open conformation. A much tighter binding is required to process individual microfibrils so a closed conformation occurs on the rearrangement of a bundle of 4 α-helices in the saddle seat. The glycine rich hinge region allows the flexibility needed for shifts between these two states. (figure.2)

Figure2:

Processing model of triple-helical and microfibrillar collagen

(a) A collagen triple helix (green) initially docks to the peptidase domain of collagenase. In the open state, the activator (dark blue) cannot interact with the substrate, with no hydrolysis occurring.

(b) Step 2, closed conformation, showing the activator HEAT repeats interacting with the triple helix, which is a prerequisite for collagen hydrolysis.

(c) Step 3, semi-opened conformation, allowing for exchange and processive degradation of all three α-chains. Once the triple helix is completely cleaved, the collagenase can relax back to the open ground state conformation, as found in our crystals, and thus complete the catalytic cycle.

(d) Collagenase with a docked collagen microfibril. The micro-fibril typically consists of five triple-helical molecules; the triple helix analogous to a is indicated in green.

(e) Step 2, closed conformation with all triple helices but one (green) being expelled from the collagenase. The microfibril wound caused by the triple-helix stripping is indicated in red.

(f) Step 3, semi-opened conformation allowing for completely processing the triple helix, indicated in green. After that, the collagenase will relax back to the open state and only then allow the remaining part of the microfibril to enter the collagenase.