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| 3D Domain swapping in cystatin C - implications for amyloidogenesis | |
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Human cystatin C (HCC) is a physiological monomeric inhibitor of
cysteine proteases. Aggregation of HCC, particularly of its L68Q
natural variant, leads to amyloid formation in the brain arteries,
and to death. The protein extracted from the amyloid fibrils is
truncated at the N-terminus. HCC was the first amyloidogenic
protein for which aggregation via 3D domain swapping was
demonstrated by X-ray crystallography. The exchange of domains
occurs after partial unfolding of the protein chains and leads
to two-fold symmetric dimers. The monomeric fold of HCC consists
of an extended beta sheet and of a long alpha helix (a) which
links strands b1 and b2. The domain that is exchanged in the
swapping act consists of the b1-a-b2 segment. Recently, we have
demonstrated that N-truncated cystatin C forms very similar
dimers using the same mechanism of domain swapping. The N-terminal
sequence, which precedes strand b1, has no influence of the
domain swapping process. Using the crystal structure of HCC as a
template, we have modified the sequences of both the wt and L68Q
HCC by introducing specific cysteine mutations designed to create
disulfide bridges that would prevent the separation of domains by
crosslinking them. The engineered proteins are indeed stabilized
by the desired S-S bonds while retaining their full potential as
protease inhibitors. Their tendency to dimerize is completely
abolished and their capacity to form amyloid fibrils under drastic
experimental conditions is greatly reduced.
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