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Neuroserpin (NS) is predominantly expressed in the brain and is the primary inhibitor of tissue plasminogen activator (tPA). NS variants are associated with the neurogenerative disease termed familial encephalopathy with neuroserpin inclusion bodies (FENIB). The disease is characterized by variable age of onset and severity. The reactive center loop (RCL) insertion-based inhibitory mechanism of NS requires a coordinated conformational change leading to a shift in the strands of the β-sheet A and movement of helix F. Strand 1A is connected to the helix F at its C terminal end and with the strand 2A at its N terminal, both these domain move for accommodating the inserting loop; therefore, a variant that influences their movement may alter the inhibition rates. A molecular dynamic simulation analysis of a H138C NS variant from strand 1A showed a large decrease in conformational fluctuations as compared with wild-type NS. H138 was mutated, expressed, purified and a native-PAGE and transmission electron microscopy (TEM) analysis showed that this variant forms large molecular weight aggregates on a slight increase in temperature. However, a circular dichroism analysis showed its secondary structure to be largely conserved. Surprisingly, its tPA inhibition activity and complex formation remain unhindered even after the site-specific labeling of H138C with Alexa fluor C5 maleimide. Further, a helix F-strand 1A (W154C-H138C) double variant still shows appreciable inhibitory activity. Increasingly, it appears that aggregation and not loss of inhibition is the more likely cause of shutter region-based variants phenotypes, indicating that hindering polymer formation using small molecules may retain inhibitory activity in pathological variants of NS. [ABSTRACT FROM AUTHOR]

Neuroserpin is a serine protease inhibitor expressed mainly in the brain and at low levels in other tissues like the kidney, testis, heart, and spinal cord. It is involved in the inhibition of tissue plasminogen activator (tPA), plasmin, and to a lesser extent, urokinase‐type plasminogen (uPA). Neuroserpin has also been shown to plays noninhibitory roles in the regulation of N‐cadherin‐mediated cell adhesion. It is involved in neuroprotection from seizure and stroke through tPA‐mediated inhibition and also through its other protease targets. Mutations in critical domains of neuroserpin lead to its polymerization and neuronal death. In this study, a novel truncated isoform of human neuroserpin was identified in the brain and liver, which was confirmed by reverse transcriptase‐PCR and DNA sequencing using exon‐specific primers. Structural characterization of novel isoform using MD simulations studies indicated that it lacks the reactive center loop (RCL) but largely maintains its secondary structure fold. The novel truncated variant was cloned, expressed, and purified. A comparative intrinsic fluorescence and 4,4′‐bis‐1‐anilino naphthalene 8‐sulfonate studies revealed a decrease in fluorescence emission intensity and a more exposed hydrophobic surface as compared to the reported isoform. However, the novel isoform has lost its ability for tPA inhibition and complex formation. The absence of RCL indicates a noninhibitory role for the truncated isoform, prompting a detailed search and identification of two smaller isoforms in the human brain. With indications of the noninhibitory role of neuroserpin, identifying novel isoforms that appear to be without the tPA recognition domain is significant. [ABSTRACT FROM AUTHOR]

Neuroserpin (NS) is an inhibitory protein of serpin super family, its shutter region variants have high propensity to aggregate leading to pathological disorders like familial encephalopathy with NS inclusion bodies (FENIB). Helix F and β-sheet A of NS participate in the tissue plasminogen activator (tPA) inhibition but the mechanism is not yet completely understood. A microsecond (μs) molecular dynamics simulation of the helix F and strand 3A variants showed predominant fluctuations in the loop connecting the strands of β-sheet A. Therefore to understand the role of helix F and strand 3A of β-sheet A, cysteine was incorporated at the position N182 in stand 3A (N182C) and position W154 (W154C) in the helix F using site-directed mutagenesis. Purified variants were further labeled with Alexa Fluor488 C 5 maleimide dye. Temperature dependent study using non-denaturing PAGE showed the formation of large aggregates of helix F variant W154C but not the strand 3A N182C variant. Interestingly tPA inhibition was found to be decreased in the labeled N182C with decreased tPA-complex formation as compared to labeled W154C NS variant. The fluorescence emission intensity of the labeled helix F variant W154C decreased in the presence of an increasing concentration of tPA, whereas an increase in emission intensity was observed in labeled strand 3A variant N182C, indicating more exposure of strand 3A and shielding of helix F. Taken together the data shows that helix F has a predominant role in the aggregation but a minor role in the inhibition mechanism. • Conformational dynamics of helix F and strand 3A of β-sheet A in neuroserpin inhibition is not easily distinguished. • Strand 3A is conformationally exposed and helix F is sheilded in the presence of tPA. • Neuroserpin helix F shows predominant effect on aggregation but not on the inhibition mechanism. [ABSTRACT FROM AUTHOR]

Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins. • It is important to understand the molecular basis of human neuroserpin variants that leads to variable clinical phenotype. • Strand 6B and helix B variant alters a hydrophobic patch that affects inhibitory activity and propensity to aggregate. • Strand 6B and helix B may link RCL (that binds tPA) with shift of strands 2A and 5A of β-sheet A for efficient inhibition. [ABSTRACT FROM AUTHOR]