Comparison of backbone dynamics of monomeric and domain‐swapped stefin A

Abstract

Three-dimensional domain swapping has been observed in increasing number of proteins and has been implicated in the initial stages of protein aggregation, including that of the cystatins. Stefin A folds as a monomer under native conditions, while under some denaturing conditions domain-swapped dimer is formed. We have determined the backbone dynamics of the monomeric and domain-swapped dimeric forms of stefin A by (15)N relaxation using a model-free approach. The overall correlation times of the molecules were determined to be 4.6 +/- 0.1 ns and 9.2 +/- 0.2 ns for the monomer and the dimer, respectively. In the monomer, decreased order parameters indicate an increased mobility for the N-terminal trunk, the first and the second binding loops. At the opposite side of the molecule, the loop connecting the alpha-helix with strand B, the beginning of strand B and the loop connecting strands C and D show increased localized mobility. In the domain-swapped dimer, a distinctive feature of the structure is the concatenation of strands B and C into a single long beta-strand. The newly formed linker region between strands B and C, which substitutes for the first binding loop in the monomer, has order parameters typical for the remainder of the beta-strands. Thus, the interaction between subunits that occurs on domain-swapping has consequences for the dynamics of the protein at long-range from the site of conformational change, where an increased rigidity in the newly formed linker region is accompanied by an increased mobility of loops remote from that site.

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?

“…This feature is not very surprising, because it is known that in solution N-and C-terminal fragments together with the loops are more Xexible than the core of a protein. This situation was observed in the case of the homologues steWn B, where the N and C termini are more Xexible in the monomer than in the dimer (Japelj et al, 2004). The higher Xexibility of the C-terminal fragment of human cystatin C can be partly explained by NMR studies of the wt and L68Q cystatin C, where the second and the Wfth -strands could be correctly assigned by using CO and C, but not C and H chemical shifts.…”

Proteomic analysis of human cervicovaginal fluid collected before preterm premature rupture of the fetal membranes

“…At sites of inflammation, cathepsins B, L and S are released during cell death and secreted by activated macrophages, causing the degradation of collagen and elastin, which may lead to ECM remodelling (Reddy et al 1995). Dimerisation of cystatin A that occurs under denaturing conditions (high temperatures 74-93 8C and low pH !2.5; Martin et al 1995, Japelj et al 2004) has been found to inactivate its inhibitory functions. We found a reduced expression of cystatin A 6-23 days before preterm PROM, which may possibly reflect a decrease in fetal membrane integrity due to an increase in unregulated proteolytic activity by cysteine proteinases.…”

Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers

“…It is well established that formation of the domain-swapped dimer of SteA is favoured by heating. 22 The dimeric form of wildtype SteA can be seen in the unheated sample (indicated by an asterisk in Figure 2(a), left-hand panel), increasing significantly following heattreatment (asterisk in Figure 2(a), right-hand panel). As predicted, this dimeric form is undetectable in both STM and STM AU1 (Figure 2(a)).…”

“…The latter change should both decrease interactions with target proteases and reduce the propensity of the scaffold to dimerize through domain swapping. 22 We refer to the engineered protein with all of these mutations as STM. An illustration of the sequence of the mutated protein with a model sequence (the AU1 peptide, see below) aligned with human StefA is shown in Figure 1(c).…”