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around the periphery of the MMP-12 active site (Fig. 6.3 ) have been implicated in
both structural interaction with solubilized, cross-linked elastin and hydrolysis of it
(Palmier et al. 2010 ). The principal exosite includes residues F202, T205, H206,
and T210 of human MMP-12 (Fig. 6.3 ), at sequence positions that distinguish MMP
subfamilies. The NMR evidence of numerous, multifaceted interactions with solu-
bilized
-elastin could explain why MMP-12 was observed bound to digested
elastin fibrils in the human aorta, even when TIMP is bound across its active site
(Curci et al. 1998 ; Liang et al. 2010 ).
Also supporting the high activity of the catalytic domain of MMP-12 appears to
be its relative rigidity and trading of some of its marginal stability for greater
activity (Liang et al. 2010 ). Those authors hypothesized that several residues distal
from the active site could modulate the stability of the catalytic domain.
a
6.2.2.4 Triple Helical Peptide Interactions Beyond the Active Site
Locations of exosite interactions with elastin appear to be relevant to triple helical
peptidase activity. MMP-12 had not been considered a collagenase. Nonethe-
less, it hydrolyzed the following triple helices of fibrillar collagens: collagen V
into peptide fragments (Fu et al. 2001 ), a triple helical peptide (THP) mimic of
the MMP-2 and -9 cleavage site in collagen V (dubbed
1(V) 436-447 fTHP)
(Bhaskaran et al. 2008 ), as well as skin collagens I and III at several sites (Taddese
et al. 2010 ).
a
1(V) 436-450 THP protected three major surfaces of MMP-12
from NMR line broadening by a small inert probe molecule: (1) the primed
(right) side of the active site cleft (center of Fig. 6.3 ), (2) the main exosite men-
tioned above (at right in Fig. 6.3 ), and (3) a distal patch on the
a
-sheet (not shown)
(Bhaskaran et al. 2008 ). Eight to ten residues around the active site cleft and at the
main exosite were found important in digestion of the triple helices from the
b
1(V)
fTHP mimic of collagen V and from labeled collagen IV, based on structurally
guided site-directed mutagenesis. All ten conservative mutations impaired rate of
catalytic turnover of the THP without harming catalytic efficiency for a linear
peptide substrate, meaning that they selectively perturb turnover of the bulky mini-
protein substrates (Palmier et al. 2010 ). Six of the lesions also impaired apparent
affinity K m for the THP substrate.
These results highlight the importance of the V-B loop region (F202, T205, and
H206 in human MMP-12) in triple-helical peptidase activity (Bhaskaran et al.
2008 ) (Palmier et al. 2010 ). This joined the prior evidence implicating the V-B
loop of MMP-1 and -8 in collagenolysis (Chung et al. 2000 ; Pelman et al. 2005 )or
triple-helical peptidase activity (Minond et al. 2006 ). Thus, the aforementioned
exosite for elastin (Fig. 6.3 ) is also important in the recognition of triple helical
substrates by MMP-12, -1, and -8. T210 at the beginning of active site helix C is
also near this patch considered to be the main exosite (Fig. 6.3 ). It was proposed that
the
a
1(V) THP bent from the MMP-12 active site across the exosite that includes
the V-B loop (Bhaskaran et al. 2008 ). This interpretation has the merits of account-
ing for (a) the protection of the exosite(s) external to the active site, (b) the
a
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