Biology Reference
In-Depth Information
types of collagenases in
C. histolyticum
: Class I collagenases (ColG) consist of
a metalloproteinase domain followed by one copy of polycystic kidney disease
domain and two copies of collagen binding domain (CBD); and class II collage-
nase (ColH) consists of a metalloproteinase domain, two copies of polycystic
kidney disease domain and one copy of CBD (Matsushita et al.
1999
). The
metalloproteinase domain has the HEYTH zinc ion binding motif, where His
415
and His
419
serveaszincligandsandGlu
447
as a third zinc ligand, making them
members of the gluzincin metalloproteinase superfamily (Jung et al.
1999
). Full-
length enzymes cleave insoluble triple helical collagens at several sites. The
enzymatic activity can also be measured with a synthetic substrate, 4-phenyla-
zobenzyloxycarbonyl-Pro-Leu~Gly-Pro-D-Arg (Pz-petide), where ~ is the cleav-
age site. The enzyme has preference for Gly in P1
0
and P3 subsites, aromatic
residues in P1, and Pro or Ala in P2 and P2
0
subsites (Van Wart and Steinbrink
1985
).
The CBDs consisting of about 110 amino acids are homologous to each other
and bind to various types of collagens by recognizing the triple helical confor-
mation of collagen, but not to heat-denatured gelatins (Toyoshima et al.
2001
;
Matsushita et al.
2001
). The CBD-deleted collagenases are not active on col-
lagens, but they have activities against gelatin and Pz-peptide substrate. Ca
2
þ
enhances the binding of CBDs to collagen at physiological concentration. In the
absence of Ca
2+
, CBD forms a
b
-sheet sandwich fold with the linker consisting of
-helix. The addition of Ca
2
þ
unwinds the linker and
anchors it to the distal side of the sandwich as a new
12 amino acids adopting an
a
-strand (Wilson et al.
2003
). However, the linker is relatively unimportant in collagen interaction. The
three conserved Tyr residues (Tyr 970, 994 and 996) are solvent exposed and
mutagenesis studies indicate that these residues are involved in collagen binding.
In particular Y994F mutation reduced the affinity to triple helical peptide (Pro-
Hyp-Gly)
8
by
b
12-fold and it is considered as the “hot-spot” (Wilson et al.
2003
). Since both (Pro-Pro-Gly)
8
and (Pro-Hyp-Gly)
8
bind to CBD tightly, it is
likely that the hydroxy group of Tyr
994
forms a hydrogen bond with the main-
chain atoms rather than to the hydroxy group of Hyp. The helix-to-sheet struc-
tural change removes the N-terminal region from the collagen binding face
(Wilson et al.
2003
).
The 3D structures of the catalytic domains of
Clostridium
collagenases have
not been solved yet. Such information, however, will assist us to further under-
stand how bacterial collagenases cleave triple helical collagen at multiple sites.
5.11 Conclusions and Perspectives
Interstitial fibrillar collagens are major components of the ECM and they play an
important role in maintaining tissues and organs by giving tensile strength. They
also are key molecules to create correct cellular environments. Although the
turnover of those fibrillar collagens in the steady state adult tissues is very slow,
