Structural comparison between wild-type and P25S human cystatin A by NMR spectroscopy. Does this mutation affect the α-helix conformation ?

Abstract

The effect of substituting Pro²⁵, located in the α-helical region of the cystatin A structure, with Ser has been studied. The structures of wild type and P25S cystatin A were determined by multidimensional NMR spectroscopy under comparable conditions. These two structures were virtually identical, and the α-helix between Glu¹⁵-Lys³⁰ exists with uninterrupted continuity, with a slight bend at residue 25. In order to characterize the possible substitution effects of Pro²⁵ with Ser on the α-helix, the chemical shifts of the amide nitrogens and protons, the generalized order parameters obtained by the analyses of the ¹⁵N-¹H relaxation data, the amide proton exchange rates, and the NOE networks among the α-helical and surrounding residues were carefully compared. None of these parameters indicated any significant static or dynamic structural differences between the α-helical regions of the wild-type and P25S cystatin A proteins. We therefore conclude that our previous structure of the wild-type cystatin A, in which the α-helix exhibited a sharp kink at Pro²⁵, must be revised. The asymmetric distribution of hydrophobic interactions between the side-chain residues of the α-helix and the rolled β-sheet surface, as revealed by NOEs, may be responsible for the slight bend of the α-helix in both variants and for the destabilized hydrogen bonding of the α-helical residues that follow Pro²⁵/Ser²⁵, as evidenced by increased amide exchange rates.