Proceedings book download - 5Z.com

Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Effect of Point Mutation in the Hinge Region on a Structure ofan Amyloidogenic Protein - Human Cystatin CMarta Orlikowska 1 , Elzbieta Jankowska 1 , Dominika Borek 2 ,Robert Kołodziejczyk 3 , Zbyszek Otwinowski 2 , Mariusz Jaskólski 3,4 ,and Aneta Szymańska 11 Department of Medicinal Chemistry, Faculty of Chemistry, University of Gdańsk, Gdańsk,Poland; 2 Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX,U.S.A. 3 Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University,Poznań, Poland; 4 Center for Biocrystallographic Research, Institute of BioorganicChemistry, Polish Academy of Sciences, Poznań, PolandIntroductionCystatins are natural inhibitors of cysteine proteases – enzymes widely distributed inanimals, plants and microorganisms. At physiological conditions hCC wt is a monomericprotein, but it was also found to be a component of massive amyloid deposits formed in abrain of patients suffering from a lethal disease called amyloid angiopathy [1].Physiologically monomeric, under crystallization conditions hCC formed the domainswapped dimer [2]. The dimerization process is probably facilitated by the presence in thehCC structure of a flexible loop L1 (55-59, QIVAG) connecting protein subdomainsundergoing the exchange process. This loop is the only part of hCC which undergoessignificant structural changes during the dimerization process which, according to theexperimental [3,4] and theoretical [5,6] studies, are driven by the conformationalconstraints attributed to the located near the top of the loop Val residue (Val57). Values ofthe angles for this residue are not optimal and that can account to propensity of theprotein to undergo the domain swapping. With the aim to check implications of greater ordecreased stability of this loop on dimerization propensity of human cystatin C and itsimpact on the overall structure of the protein, we designed and obtained hCC L1 variantswith Val57 residue replaced by Asp, Asn ( -turn stabilization) or Pro (turn broadening). Allproteins were subjected to dimerization studies and their structure was determined bymeans of crystallography.Results and DiscussionProtein state in solution was examined by gel-filtration on Superdex 75 column(GE Healthcare) (Figure1). The retention times of the aspartic acid and asparagine mutantswere similar to that of the wild type protein whereas the proline mutant eluted from thecolumn as two peaks (monomer/dimer equilibrium). In vitro dimerization experimentsshowed that, unlike the wild type protein, two of the studied variants of hCC (V57D andV57N) did not undergo dimerization induced by growing concentration of destabilizingagents, e.g guanidinum chloride whereas for thepredominantly dimeric proline variant the fractionof the dimer further increased with incubation time[7]. These results confirmed predicted stabilizingeffect of V57N and V57D mutations anddestabilizing effect of the proline substitution.The crystal structures of V57D, V57N andV57P variants at the resolution 3.0, 2.04 and2.25Å respectively, reveal the typical cystatin foldwith a five-stranded antiparallel -sheet wrappedFig. 1. Gel filtration chromatogramsof wt hCC and its variant.around an -helix (Figure 2). Crystallization ofV57N resulted in the monomeric structure [8].This result is very interesting considering thelimited scope of the structural intervention in thismutant. The single point mutation stabilizing theL1 loop was sufficient to preserve human cystatinC molecule in a monomeric form. Mutant V57Dunder crystallization conditions (0.1 M Imidazole280