A hydrophobic patch surrounding Trp154 in human neuroserpin controls the helix F dynamics with implications in inhibition and aggregation

Abstract

Human Neuroserpin (NS) a member of serine protease inhibitor (serpin) superfamily is expressed throughout the nervous system but more specifically at the later stages of neuronal cell development [1][2][3] . NS inhibits serine protease tissue-type plasminogen activator (tPA), which has been reported to play critical role in memory, synaptic plasticity and brain development 2,4,5 . Lack of NS activity or its polymerisation can directly lead to dementia and epilepsy 6,7 . NS with specific point mutations like S49P, S52R, H338R, G392E and G392R have been recognized to cause familial encephalopathy with neuroserpin inclusion bodies (FENIB) [6][7][8][9][10] . These mutations are located around the shutter region and are believed to cause opening of β -sheet A which leads to the formation of polymers via loop-sheet polymerisation mechanism 8,9,11 . Deficiency in other inhibitory serpins like α 1 -antitrypsin (AAT), α 1 -antichymotrypsin (AAC), C1-inhibitor, plasminogen activator inhibitor-1 (PAI-1) and antithrombin (AT) may result in pathological disorders like emphysema/cirrhosis, chronic obstructive bronchitis, angio-edema, bleeding disorder and thrombosis respectively [12][13][14][15][16] . These pathological disorders are collectively termed as 'serpinopathies'9 . Under normal conditions the circulatory inhibitory form of wild type (WT) serpin exists in a metastable conformation [17][18][19] . However, 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 4A 20 . During this process the protease remains covalently trapped, almost irreversibly as an acyl enzyme intermediate 3 .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 A 21,22 .

Neuroserpin: structure, function, physiology and pathology

“…Helix F and β-sheet A were found to be crucial, since restoring the aminoacidic composition to the serpin consensus sequence increased the stability and reduced the polymerization tendency of neuroserpin [ 24 ]. In agreement with this, disrupting the interaction between helix F and β-sheet A led to a higher polymerisation rate or a tendency to precipitate [ 27 ]. Finally, mutations in helix B and β-sheet B reduced the propensity to polymerization but promoted the transition to the latent conformation [ 57 ].…”

“…The region including helix F and strand 1 of β-sheet A also presents some aminoacidic substitutions when compared to the serpin consensus sequence: Asn162Gly, Leu162Lys and Val63Ile, which decrease the stability of helix F increasing the tendency to adopt the latent conformation, as also observed for plasminogen activator inhibitor 1 (PAI-1) and tengpin [ 25 , 26 ]. Alterations of helix F dynamics can also reduce inhibitory activity and increase polymerisation [ 27 ]. Finally, the packing of β-sheet A is particularly tight thanks to an extra interaction between His338 and Ser340, which in other serpins is usually an alanine.…”

Detection of truncated isoforms of human neuroserpin lacking the reactive center loop: Implications in noninhibitory role

“…The delicate balance between neuroserpin and tPA reduces the neurotoxicity in the neuron, while the accumulation of neuroserpin variants can lead to neurodegeneration. Due to its mechanism of action, which requires large‐scale conformational changes during tPA inhibition, neuroserpin is prone to aggregation 29 . Several natural variants of neuroserpin are directly implicated in giving rise to dementia/epilepsy due to the formation of neuroserpin aggregates, with variable severity and age‐onset 35 .…”

“…Due to its mechanism of action, which requires large-scale conformational changes during tPA inhibition, neuroserpin is prone to aggregation. 29 Several natural variants of neuroserpin are directly implicated in giving rise to dementia/epilepsy due to the formation of neuroserpin aggregates, with variable severity and ageonset. 35 Interestingly, an increasing number of studies point to a number of tPA-independent roles of neuroserpin, with a total lack of understanding of its molecular basis.…”

“…For the structural characterization of the HNS-T isoform, site-directed mutagenesis (SDM) was carried out using plasmid, pET-28b(+), carrying NS gene with N-terminal His-tag. 29 A stop codon was introduced by SDM at a specific location to mimic the novel truncated isoform of neuroserpin identified in this study. The amino acid sequence of SDM generated variant and HNS-T are almost identical.…”