The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Abstract

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three-dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the beta2 and beta3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the beta2-L1-beta3 (His43-Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the beta2-L1-beta3 peptide, in which the Val57 residue was replaced by residues promoting beta-turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the beta-hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation.

Influence of point mutations on the stability, dimerization, and oligomerization of human cystatin C and its L68Q variant

“…Comparison of its sequence with the data for other cystatins available currently in the PDB database, and further analysis in the context of the torsional φ and ψ angle values revealed, that there is the amino acid residue displaying strained conformation, namely valine in position 57, located near the apex of the loop (Rodziewicz-Motowidło et al, 2009; Figure 1A). These tensions may be important for the biological activity of cystatin C, but they may also account for its increased dynamics, especially in the absence of the target protease.…”

Hinge-loop mutation can be used to control 3D domain swapping and amyloidogenesis of human cystatin C

“…Experimental (Engh et al, 1993;Martin et al, 1995) and theoretical (Dehouck et al, 2003;Rodziewicz-Motowidło et al, 2009) studies revealed that the L1 region of cystatin C is conformationally unstable with the largest component of instability related to the residue V57, located at the apex of the loop. The values of w angles for this residue are not optimal (Rodziewicz-Motowidło et al, 2009), what can explain propensity of the protein to undergo domain swapping.…”

“…The values of w angles for this residue are not optimal (Rodziewicz-Motowidło et al, 2009), what can explain propensity of the protein to undergo domain swapping. It agrees with the literature reports linking presence of flexible hinge loops in a protein structure with its ability to swap domains (Ding et al, 2006).…”

Application of amide hydrogen/deuterium exchange mass spectrometry for epitope mapping in human cystatin C

“…During domain swapping process, conformational changes concerning the flexible loops as well as β-turns occur (Szymańska et al 2009). The part of the protein that is significantly changed during dimerization is loop L1 which disappears in the dimeric hCC structure (Rodziewicz-Motowidło et al 2009). This region, with its lack of conformational stability, is considered to be the molecular “spring” facilitating the domain exchange (Rodziewicz-Motowidło et al 2009).…”