Biomedical Engineering Reference
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specificity. It is therefore likely that SMIPPs do not function by competing for
substrates with host proteases that play a role in host defense mechanisms. It is
also likely that all members of the SMIPP family have lost the ability to bind
substrates by a known mechanism, and instead have evolved alternative
binding activities.
10.6.1 Putative Alternative Binding Sites
Consistent with their large structural differences, the shape and electrostatic
properties of their active sites are strikingly contrasting (Figure 10.4A and B).
Although the structural changes observed in SMIPP-S-I1 preclude peptide or
protein substrate binding in a serine protease-like fashion (Figure 10.2), its
'active site' still resembles a deep groove (Figure 10.4A). Further, the surface of
the groove is highly negatively charged (with the charge extending beyond the
groove), suggestive of a binding function. In contrast, the equivalent region in
SMIPP-S-D1 is relatively shallow and, with the exception of two negatively
charged small pockets, is relatively neutral.
10.6.2 Sequence Conservation in the SMIPP Family
In search of further clues to function, the sequence conservation within the
SMIPP family was mapped onto both structures (Figure 10.4C-E). It has been
shown that sequence conservation of surface residues is a robust indicator of
functional sites.
19,20
The low sequence conservation within the SMIPP family is
revealed clearly: the highest conservation is found within the core of the
molecule (with the exception of the catalytic triad; Figure 10.4C and D),
whereas the surrounding, more surface-exposed regions show little if any
conservation. This is consistent with the structural differences between the
Figure 10.3
Neighbour-joining bootstrap tree (500 replicates) illustrating the inferred
evolutionary relationships between the SMIPPs generated in MEGA4
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with maximum-likelihood branch lengths inferred using the WAG amino
acid matrix in PAML4.1.
22
The tree is rooted at the vertebrate-arthropod
divergence. Whole numbers at the nodes indicate the level of bootstrap
support,
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and branches with less than 80% support have been collapsed.
Italicised fractions at the nodes indicate the lowest fractional sequence
identity among descendant sequences. Sequence names with an asterisk
represent incomplete sequences; a plus symbol denotes proteases with an
intact catalytic triad (HDS). On the right-hand side of each clade, muta-
tions of the catalytic triad residues for that clade are shown as horizontal
triplets, corresponding with the H63, D107, and S200 positions, respec-
tively (human trypsin numbering). Dashed lines indicate lineages for which
structural divergence is predicted:
รพ
indicates an insertion or acquisition,
and D indicates a deletion or loss. The scale bar shows a distance of 0.2
mutations per amino acid residue. The outgroup sequences are D. pter-
onyssinus Der p 3 (accession DERP3_DERPT), D. melanogaster gamma
trypsin (TRYG_DROME), H. sapiens trypsin 2 (TRY2_HUMAN), and
D. rerio trypsin (NP_571783). The tree was drawn using MEGA4.
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