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.

中文翻译：

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神经丝氨酸蛋白酶抑制剂抑制期间链 6B 和螺旋 B 的变化：对临床表型严重程度的影响。


Neuroserpin (NS) 主要在大脑中表达，并抑制组织型纤溶酶原激活剂 (tPA)，对大脑发育和记忆有影响。 NS 6B 链和 B 螺旋中导致相对轻度至重度癫痫（和/或痴呆）的病理变异的构象变化的性质在很大程度上仍然难以捉摸。使用野生型 (WT) NS、链 6B 和螺旋 B 变体进行的 MD 模拟表明，该区域的取代会影响链 5B、5A 和反应中心环的构象。因此，我们设计了 6B 链（I46D 和 F48S）和 B 螺旋（A54F、L55A 和 L55P）中的 NS 变体，以研究它们在 tPA 抑制机制和聚集倾向中的作用。相互作用分析显示，在 I46D 和 F48S 中，以链 5B、6B 和螺旋 B 为中心的疏水斑块受到干扰，但在 A54F、L55A、L55P 和 WT NS 中则没有。纯化的 I46D、F48S 和 L55P 变体显示荧光发射强度降低，但具有相似的 α 螺旋含量，但 A54F 和 L55A 的结果与 WT NS 相当。 tPA 抑制分析显示对 A54F 和 L55A 变体与 tPA-NS 复合物形成的边际效应。相比之下，I46D、F48S 和 L55P 变体显示 tPA 抑制大量减少，且没有 tPA-NS 复合物形成。聚合条件下的天然 PAGE 分析显示 I46D、F48S 和 L55P 迅速转化为潜在构象，但 A54F 和 L55A 变体则不然。识别这些新的构象变化将有助于理解快门区域 NS 变体和其他丝氨酸蛋白酶抑制剂的可变临床表型。