The crystal structure of human cystatin C, a protein with amyloidogenic properties and a potent inhibitor of cysteine proteases, reveals how the protein refolds to produce very tight two-fold symmetric dimers while retaining the secondary structure of the monomeric form. The dimerization occurs through three-dimensional domain swapping, a mechanism for forming oligomeric proteins. The reconstituted monomer-like domains are similar to chicken cystatin except for one inhibitory loop that unfolds to form the 'open interface' of the dimer. The structure explains the tendency of human cystatin C to dimerize and suggests a mechanism for its aggregation in the brain arteries of elderly people with amyloid angiopathy. A more severe 'conformational disease' is associated with the L68Q mutant of human cystatin C, which causes massive amyloidosis, cerebral hemorrhage and death in young adults. The structure of the three-dimensional domain-swapped dimers shows how the L68Q mutation destabilizes the monomers and makes the partially unfolded intermediate less unstable. Higher aggregates may arise through the three-dimensional domain-swapping mechanism occurring in an open-ended fashion in which partially unfolded molecules are linked into infinite chains.

Stability constants and ligand donor sets of the copper(II) complexes of the NH2-29-56(L1)(AA30GKTKEGVLYV40GSKTKEGVVH50GVATVA56-NH2), NH2-M29-D30-56(L2) and Ac-M29-D30-56(L3) fragments of alpha-synuclein were determined in aqueous solution for 1 : 1 metal-to-ligand molar ratio in the pH range 2.5-10.5. The tyrosine residue in the 39th position of the alpha-synuclein fragments does not take part in the coordination of the metal ion. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the L3 fragment compared to that of the L2 peptide. When the amino group is blocked (L3) the imidazole nitrogen of the histidine residue acts as an anchoring site and at higher pH the 3N {N(Im),2N-} and 4N {N(Im),3N-} complexes are formed. The L1 peptide at physiological pH forms in equilibrium 3N {NH2,N-,CO,N(Im)} and 4N {NH2,2N-,N(Im)} complexes. For the L2 peptide the coordination of the copper(II) ions starts from the N-terminal Met residue and with increasing of pH the Asp residue in second position of amino acid sequence coordinates and stabilizes significantly the 2N complex as a result of chelation through the beta-carboxylate group. At physiological pH the 3N {NH2,N-,beta-COO-,N(Im)} coordination mode dominates. At pH above 6 the results for the L2 fragment suggest the formation of 3N and 4N complexes (in equatorial plane) and the involvement of the lateral NH2 group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra sigma (epsilon-NH2-Lys) --> Cu(II) charge transfer transition is observed. The stability constants for the L2 fragment of alpha-synuclein of the 4N {NH2,2N-,N(Im)} and {NH2,3N-} complexes are higher about 1.5 and 0.7 orders of magnitude, respectively, by comparison to those of the L1 peptide. This increase may be explained by the involvement of the epsilon-NH2 group of Lys residue in the coordination sphere of metal ion.

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Human cystatin C (HCC) is a protease inhibitor with a propensity to form beta-amyloid (Abeta)-like fibrils and to coassociate with amyloidogenic proteins. Recently, a specific interaction between HCC and Abeta has been found. Here, we report the identification of the Abeta and HCC binding epitopes in the Abeta-HCC complex, using a combination of selective proteolytic excision and high resolution mass spectrometry. Proteolytic excision of Abeta(1-40) on sepharose-immobilized HCC and MALDI-MS identified the epitope Abeta(17-28). On immobilized Abeta(1-40), affinity MS of HCC fragments identified a specific C-terminal epitope, HCC(101-117). Binding specificities of both epitopes were ascertained by ELISA and surface plasmon resonance and by direct electrospray MS of the HCC-Abeta epitope peptide complexes. A structure model of the HCC-Abeta complex by molecular docking simulation showed full agreement with the identified Abeta and HCC epitopes. Inhibition studies in vitro revealed Abeta-fibril inhibiting activity of the HCC(101-117)-epitope. The Abeta-HCC interacting epitopes provide lead structures of neuroprotective inhibitors for AD and HCC amyloidosis therapy.