title: Structural characterization of V57D and V57P mutants of human cystatin C, an amyloidogenic protein.
abstract: Wild-type human cystatin C (hCC wt) is a low-molecular-mass protein (120 amino-acid residues, 13,343 Da) that is found in all nucleated cells. Physiologically, it functions as a potent regulator of cysteine protease activity. While the biologically active hCC wt is a monomeric protein, all crystallization efforts to date have resulted in a three-dimensional domain-swapped dimeric structure. In the recently published structure of a mutated hCC, the monomeric fold was preserved by a stabilization of the conformationally constrained loop L1 caused by a single amino-acid substitution: Val57Asn. Additional hCC mutants were obtained in order to elucidate the relationship between the stability of the L1 loop and the propensity of human cystatin C to dimerize. In one mutant Val57 was substituted by an aspartic acid residue, which is favoured in β-turns, and in the second mutant proline, a residue known for broadening turns, was substituted for the same Val57. Here, 2.26 and 3.0 Å resolution crystal structures of the V57D andV57P mutants of hCC are reported and their dimeric architecture is discussed in terms of the stabilization and destabilization effects of the introduced mutations.
authors.affiliation: Department of Medicinal Chemistry, Faculty of Chemistry, University of Gdansk, Gdansk, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Orlikowska
authors.forename: Aneta
authors.initials: A
authors.lastname: Szymańska
authors.forename: Dominika
authors.initials: D
authors.lastname: Borek
authors.forename: Zbyszek
authors.initials: Z
authors.lastname: Otwinowski
authors.forename: Piotr
authors.initials: P
authors.lastname: Skowron
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Jankowska
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D058227:Amyloidogenic Proteins / Q000737:chemistry* / Q000235:genetics*; D001224:Aspartic Acid / Q000235:genetics; D018360:Crystallography, X-Ray; D055316:Cystatin C / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D014633:Valine / Q000235:genetics
chemical_list: D058227:Amyloidogenic Proteins; D055316:Cystatin C; D001224:Aspartic Acid; D011392:Proline; D014633:Valine
keywords: amyloid; cysteine protease inhibitors; human cystatin C; single-point mutations