Hinge-loop mutation can be used to control domain swapping of human cystatin C

Abstract

Poster Sessions C310 exogenous recombinant 'truncated' Galectin-3 (analogous to the MMPprocessed form), but not full length Galectin-3, dramatically increases migration of the human breast cancer cell line BT-549. These results suggest that the MMP-cleaved Galectin-3 and the resulting structural changes are responsible for pro-metastatic properties of Galectin-3. We have obtained crystallographic data for CRD of Galectin-3 in complex with a pentasaccharide to which Galectin-3 has higher binding affinity compared to other galectin family members. This structural information may be utilized in the design of Galectin-3 specific inhibitors targeting the carbohydrate-binding site. We have also explored the structural differences resulting from MMP cleavage of Galectin-3 using SAXS.[1] F.T. Liu and G.A. Rabinovich, Nat Rev Cancer, 2005, 5, 29-41 Gdańsk, Gdańsk, (Poland). E-mail: martaorlikowska07@gmail.com Human cystatin C (hCC) is a low molecular mass protein (120 amino acid residues, 13,343 Da) that belongs to a family of single chain, reversible inhibitors of papain-like (C1 family) and legumainrelated (C13 family) cysteine proteases [1]. In pathophysiological processes, which nature of is not understood, hCC is codeposited in the amyloid plaques of Alzheimer's disease or Down's syndrome. The amyloidogenic properties of HCC are greatly increased in a naturally occurring L68Q variant, resulting in fatal cerebral amyloid angiopathy in early adult life [2]. At physiological conditions wild-type hCC is a monomeric protein, but under crystallization conditions (pH 4.8) forms a domain-swapped dimer [3]. The dimerization process is facilitated by the presence in the hCC structure of a flexible region created by the loop L1 (55-59, QIVAG) connecting protein subdomains undergoing the exchange process. This loop is the only part of hCC which undergoes significant structural changes during the dimerization process and, according to experimental [4], [5] and theoretical [6], [7] studies, these changes are driven by the conformational constraints attributed to the located near the top of the loop Val residue (Val57 for hCC).With the aim to check implications of grater or decreased stability of this loop on dimerization and aggregation propensity of human cystatin C, we designed and constructed hCC L1 mutants with Val57 residue replaced by Asp, Asn [8], Gly (residues favored in this position of β-turns) or Pro, respectively. By applying this rational mutagenesis approach we were able to obtain hCC variants stable in the monomeric form both in solution and in the crystal (V57G, Figure 1), monomeric in solution but dimeric in the crystal (V57D) and dimeric in solution and oligomeric in the crystal (V57P). The results of structural studies of hCC L1 mutants will be presented.

An Overview about the Relationship between Cystatin C and Inflammatory Diseases

“…It has a molecular mass of 13 kD, an isoelectric point of 9.3, and contains 122 amino acids. It is an intrinsic inhibitor of cysteine proteases such as cathepsin B, elastase, and please (Figure 1) [1].…”