PDBsum entry 3fgq

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Hydrolase inhibitor PDB id
Protein chains
374 a.a. *
GOL ×2
Waters ×296
* Residue conservation analysis
PDB id:
Name: Hydrolase inhibitor
Title: Crystal structure of native human neuroserpin
Structure: Neuroserpin. Chain: a, b. Fragment: unp residues 17-400. Synonym: serpin i1, protease inhibitor 12. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.
2.09Å     R-factor:   0.176     R-free:   0.226
Authors: S.Takehara,X.Yang,B.Mikami,M.Onda
Key ref:
S.Takehara et al. (2009). The 2.1-A crystal structure of native neuroserpin reveals unique structural elements that contribute to conformational instability. J Mol Biol, 388, 11-20. PubMed id: 19285087 DOI: 10.1016/j.jmb.2009.03.007
08-Dec-08     Release date:   28-Apr-09    
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Protein chains
Pfam   ArchSchema ?
Q99574  (NEUS_HUMAN) -  Neuroserpin
410 a.a.
374 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   3 terms 
  Biological process     cell death   7 terms 
  Biochemical function     peptidase inhibitor activity     2 terms  


DOI no: 10.1016/j.jmb.2009.03.007 J Mol Biol 388:11-20 (2009)
PubMed id: 19285087  
The 2.1-A crystal structure of native neuroserpin reveals unique structural elements that contribute to conformational instability.
S.Takehara, M.Onda, J.Zhang, M.Nishiyama, X.Yang, B.Mikami, D.A.Lomas.
Neuroserpin is a selective inhibitor of tissue-type plasminogen activator (tPA) that plays an important role in neuronal plasticity, memory, and learning. We report here the crystal structure of native human neuroserpin at 2.1 A resolution. The structure has a helical reactive center loop and an omega loop between strands 1B and 2B. The omega loop contributes to the inhibition of tPA, as deletion of this motif reduced the association rate constant with tPA by threefold but had no effect on the kinetics of interaction with urokinase. Point mutations in neuroserpin cause the formation of ordered intracellular polymers that underlie dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type neuroserpin is also unstable and readily forms polymers under near-physiological conditions in vitro. This is, in part, due to the substitution of a conserved alanine for serine at position 340. The replacement of Ser340 by Ala increased the melting temperature by 3 degrees C and reduced polymerization as compared to wild-type neuroserpin. Similarly, neuroserpin has Asn-Leu-Val at the end of helix F and thus differs markedly from the Gly-X-Ile consensus sequence of the serpins. Restoration of these amino acids to the consensus sequence increased thermal stability and reduced the polymerization of neuroserpin and its transition to the latent conformer. Moreover, introduction of the consensus sequence into S49P neuroserpin that causes FENIB increased the stability and inhibitory activity of the mutant, as well as blocked polymerization and increased the yield of protein during refolding. These data provide a molecular explanation for the inherent instability of neuroserpin and the effect of point mutations that underlie the dementia FENIB.
  Selected figure(s)  
Figure 1.
Fig. 1. (a) Sequence alignment of human neuroserpin (NS), α[1]-antitrypsin (α[1]-AT), antithrombin (ANT), plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2), and chicken ovalbumin (OVA). Sequence numbering is based on neuroserpin, and shading indicates conserved residues within the serpin superfamily.^24 The following residues are highlighted in neuroserpin: Ser49, Ser52, Asn182, and His338 (open circles); Ser111 and Ala135 (closed triangles); Trp154 (open triangle); Asn161, Leu162, and Val163 (asterisks); from Gly231 to Ala235 (crosses); Ser340 (closed circle). “h” and “s” represent helix and strand, respectively. (b) The crystal structure of native human neuroserpin. The strands of β-sheet A are shown in green along with the reactive center loop (red), the P1 residue (R362; magenta), helix F (cyan), the omega loop between strands 1B and 2B (orange), and the three-residue C-terminal extension (yellow). Blue spheres represent the residues that are implicated in the instability and polymerization of wild-type neuroserpin. The shutter region is depicted with a red broken circle. (c) Stereo view of the electron density map (blue 2F[o] − F[c] contoured at 1.2σ) of the residues in the 3[10] helix in the reactive center loop (Ser361-Leu366; yellow) and the other two neuroserpin molecules (cyan and agenda) that have contacts with the residues in the reactive center loop. (d) Complex formation between tPA and wild-type (WT) or Δloop neuroserpin. Neuroserpin (3 mM) was incubated with 1.5 mM tPA at 25 °C for 0, 1, or 30 min in a reaction buffer [50 mM Hepes, 150 mM NaCl, and 0.01% dodecylmaltoside (pH 7.4)], and the sample was analyzed by SDS-PAGE. The numbers indicated above each lane represent the incubation time (in minutes). Cpx, N, and Cl represent the complex, intact neuroserpin, and cleaved neuroserpin, respectively.
Figure 2.
Fig. 2. (a) Electrostatic surface of neuroserpin (blue, positive; red, negative). The schematics are oriented as in Fig. 1b. (b) Superposition of human native neuroserpin (green) and mouse-cleaved neuroserpin (red; 1JJO).^16 Helices B–F are marked with black (human) or yellow (mouse) letters. β-Sheet A is numbered in black (human) or yellow (mouse). The P14 residue (Ser349) is depicted with a sphere. (c) Electron density map (blue 2F[o] − F[c] contoured at 1.2σ) and omit map (purple F[o] − F[c] contoured at 3σ) of the five residues in the hydrogen bond network in the shutter region calculated following the substitution of Asn182 by Ala. Hydrogen bonds are depicted with a dotted red line. (d) Schematic view showing the interactions between Trp/Tyr in helix F and strand 1A/2A. α[1]-Antitrypsin (orange; 1QLP)^23 and ovalbumin (magenta; 1OVA)^26 are superimposed on neuroserpin (green). The following residues are shown in stick form: Ser111, Ala135, and Trp154 in neuroserpin; Glu141 and Tyr160 in α[1]-antitrypsin; and Arg117 and Trp160 in ovalbumin. The hydrogen bond between Tyr160 in helix F and Glu141 in strand 1A of α[1]-antitrypsin is depicted with a red dotted line.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 388, 11-20) copyright 2009.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19953505 D.Belorgey, P.Hägglöf, M.Onda, and D.A.Lomas (2010).
pH-dependent stability of neuroserpin is mediated by histidines 119 and 138; implications for the control of beta-sheet A and polymerization.
  Protein Sci, 19, 220-228.  
21081089 S.Ricagno, M.Pezzullo, A.Barbiroli, M.Manno, M.Levantino, M.G.Santangelo, F.Bonomi, and M.Bolognesi (2010).
Two latent and two hyperstable polymeric forms of human neuroserpin.
  Biophys J, 99, 3402-3411.  
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