Biomedical Engineering Reference
In-Depth Information
10.2.2 Structural Diversity Within the Active Site and
Throughout the Molecules
In comparison to each other and with trypsin, the SMIPPs display relatively
large (
0.4 nm) backbone shifts extending throughout the structure, parti-
cularly in loop regions (residues 20-26, 131-140, and 201-206, corresponding
to residues 35-40, 142-153, and 218-223 in chymotrypsin numbering).
However, the core interiors of the molecules are relatively well conserved,
and the positions of the catalytic triad (or equivalent residues) are main-
tained (Figure 10.1A and B). The relatively large structural divergence
between the two SMIPPs, and also in comparison to trypsin, reflects the low
sequence identities between the SMIPPs and also with the mammalian
enzyme (Figure 10.1A). Indeed, the sequence divergence within the SMIPP
family is suciently large to imply that the majority of other SMIPPS also
display large, unpredictable structural variations outside the core of the
In both structures, the S1 subsite is blocked by a conserved tyrosine (Y200,
215 in chymotrypsin numbering), thus occluding substrate access to this vital
part of the active site (Figure 10.1A). Structural shifts in this region are rela-
tively large, particularly in the loop (201-206, 218-223 in chymotrypsin num-
bering) that contains Cys220 in trypsin. Since all SMIPPs lack the Cys191 and
Cys220 residues that form a conserved disulfide bond in the serine protease
family, it is likely that all display similarly large loop shifts resulting in a
pronounced structural rearrangement at the S1 subsite.
Interestingly, although SMIPPs do not contain zymogen sequences, 12 pre-
dictions show signal peptidase cleavage at the site usually expected after clea-
vage of a zymogen, for every SMIPP known. In addition, they have several
structural characteristics in common with the zymogen form. First, some of the
regions showing structural differences correspond to the 'zymogen activation
domain' of chymotrypsinogen (i.e., the 147-loop, 180-loop, and 220-loop, in
chymotrypsin numbering). Second, whereas in SMIPP-S-D1 the oxyanion hole
is properly formed, it is slightly distorted in SMIPP-S-I1 (though this is rela-
tively small compared to the disruption seen in chymotrypsinogen). Third, in
SMIPP-S-I1 the N-terminus incorporation has not occurred, resulting in a
zymogen-like conformation of the N-terminus, but not the 'zymogen-triad'
seen in chymotrypsinogen (this requires a greater rearrangement of the oxy-
anion hole).
Superimposing SMIPP structures with a trypsin-protein inhibitor complex
(2PTC) reveals that the structural changes are so large that formation of a
complex with a protein substrate would be impeded without extensive struc-
tural rearrangement (Figure 10.2). It has been suggested that SMIPPs may
function by binding and protecting target substrates from cleavage by host
immune proteases, thus preventing the host from mounting an effective
immune challenge. 13 This hypothesis is clearly incompatible with the structural
data, but nevertheless it is interesting to ask next whether SMIPPs are able to
bind peptide substrates.
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