Abstract
Neuroserpin (NS) mediated inhibition of tissue-type plasminogen activator (tPA) is important for brain development, synapse formation and memory. Aberrations in helix F and β-sheet A movement during inhibition can directly lead to epilepsy or dementia. Conserved W154 residue in a hydrophobic patch between helix F and β-sheet A is ideally placed to control their movement during inhibition. Molecular Dynamics (MD) simulation on wild type (WT) NS and its two variants (W154A and W154P) demonstrated partial deformation in helix F and conformational differences in strands 1A and 2A only in W154P. A fluorescence and Circular Dichroism (CD) analysis with purified W154 variants revealed a significant red-shift and an increase in α-helical content in W154P as compared to W154A and WT NS. Kinetics of tPA inhibition showed a decline in association rates (ka) for W154A as compared to WT NS with indication of complex formation. Appearance of cleaved without complex formation in W154P indicates that the variant acts as substrate due to conformational misfolding around helix F. Both the variants however showed increased rate of aggregation as compared to WT NS. The hydrophobic patch identified in this study may have importance in helix F dynamics of NS.
Highlights
Human Neuroserpin (NS) a member of serine protease inhibitor superfamily is expressed throughout the nervous system but at the later stages of neuronal cell development[1,2,3]
We observed that W154P variant demonstrated greater mobility in helix F, loop and strand 1A region; the mobility was reduced in strand 2A than the W154 to alanine (W154A) variant (Fig. 2)
Both the loop connecting helix F to strand 3A and 1A were found to be shifted in case of W154P variant thereby, resulting in an increased conformational flexibility (Figs 2 and 3)
Summary
Human Neuroserpin (NS) a member of serine protease inhibitor (serpin) superfamily is expressed throughout the nervous system but at the later stages of neuronal cell development[1,2,3]. During its inhibitory action on serine proteases it acquires a stable conformation upon cleavage of the reactive center loop (RCL) and insertion into the β-sheet A as an additional strand 4A20. As a result the conformational dynamics of serpin inhibition mechanism makes it prone to polymer formation The latter is attributed to the large scale movements in RCL, helix F and strands of β-sheet A21,22. The structure revealed that the loop connecting helix F to strand 3A of β-sheet A partially fills the space between the sheet A27, leading to loss of its activity In view of these facts, it is likely that NS inhibition of tPA would require the movement of the helix F, the residues contributing to this movement and their exact role in the mechanism that links inhibition and polymerisation still remains unclear. The results of the present study demonstrated that hydrophobic interactions between W154 of helix F and β-sheet A are important and needs to be thoroughly tested for helix F movement during loop insertion and in preventing aggregate formation during inhibition
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