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PDBsum entry 3jz1

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protein ligands metals Protein-protein interface(s) links
Hydrolase PDB id
3jz1
Jmol
Contents
Protein chains
30 a.a. *
248 a.a. *
Ligands
NO3 ×2
NAG
GOL
Metals
_NA ×2
Waters ×214
* Residue conservation analysis
PDB id:
3jz1
Name: Hydrolase
Title: Crystal structure of human thrombin mutant n143p in e:na+ fo
Structure: Thrombin light chain. Chain: a. Engineered: yes. Mutation: yes. Thrombin heavy chain. Chain: b. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606. Gene: f2. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_taxid: 10029
Resolution:
1.60Å     R-factor:   0.203     R-free:   0.216
Authors: W.Niu,Z.Chen,L.A.Bush-Pelc,A.Bah,P.S.Gandhi,E.Di Cera
Key ref:
W.Niu et al. (2009). The mutant N143P reveals how Na+ activates thrombin. J Biol Chem, 284, 36175-36185. PubMed id: 19846563 DOI: 10.1074/jbc.M109.069500
Date:
22-Sep-09     Release date:   20-Oct-09    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chain
Pfam   ArchSchema ?
P00734  (THRB_HUMAN) -  Prothrombin
Seq:
Struc:
 
Seq:
Struc:
622 a.a.
30 a.a.
Protein chain
Pfam   ArchSchema ?
P00734  (THRB_HUMAN) -  Prothrombin
Seq:
Struc:
 
Seq:
Struc:
622 a.a.
248 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: Chains A, B: E.C.3.4.21.5  - Thrombin.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   1 term 
  Biological process     blood coagulation   2 terms 
  Biochemical function     catalytic activity     3 terms  

 

 
DOI no: 10.1074/jbc.M109.069500 J Biol Chem 284:36175-36185 (2009)
PubMed id: 19846563  
 
 
The mutant N143P reveals how Na+ activates thrombin.
W.Niu, Z.Chen, L.A.Bush-Pelc, A.Bah, P.S.Gandhi, E.Di Cera.
 
  ABSTRACT  
 
The molecular mechanism of thrombin activation by Na+ remains elusive. Its kinetic formulation requires extension of the classical Botts-Morales theory for the action of a modifier on an enzyme to correctly account for the contribution of the E*, E and E:Na+ forms. The extended scheme establishes that analysis of kcat unequivocally identifies allosteric transduction of Na+ binding into enhanced catalytic activity. The thrombin mutant N143P features no Na+-dependent enhancement of kcat, yet binds Na+ with an affinity comparable to that of wild-type. Crystal structures of the mutant in the presence and absence of Na+ confirm that Pro143 abrogates the important H-bond between the backbone N atom of residue 143 and the carbonyl O atom of Glu192, which in turn controls the orientation of the Glu192-Gly193 peptide bond and the correct architecture of the oxyanion hole. We conclude that Na+ activates thrombin by securing the correct orientation of the Glu192-Gly193 peptide bond, which is likely flipped in the absence of cation. Absolute conservation of the 143-192 H-bond in trypsin-like proteases and the importance of the oxyanion hole in protease function suggest that this mechanism of Na+ activation is present in all Na+-activated trypsin-like proteases.
 
  Selected figure(s)  
 
Figure 2.
Na^+ binding to thrombin wild-type and N143P. A, kinetic traces of Na^+ binding to wild-type thrombin in the 0–250 ms time scale. Shown are the traces obtained at 0 (open circles) and 50 mm Na^+ in the absence (black circles) or presence (gray circles) of 1 mm benzamidine. Notice how the binding of Na^+ obeys a two-step mechanism, with a fast phase completed within the dead time (<0.5 ms) of the spectrometer, followed by a single-exponential slow phase. The presence of benzamidine abolishes the slow phase and yields a rapid phase whose amplitude is comparable to that observed in the absence of benzamidine. Experimental conditions are: 50 nm thrombin, 5 mm Tris, 0.1% PEG 8000, pH 8.0, at 15 °C. The [Na^+] was changed by keeping the ionic strength constant at 400 mm with ChCl. Continuous lines were drawn using the expression b − a exp(−k[obs]t) with best-fit parameter values: open circles, a = 0 ± 0 V, k[obs] = 0 ± 0 s^−1, b = 8.11 ± 0.01 V; black circles, a = 0.27 ± 0.02 V, k[obs] = 130 ± 10 s^−1, b = 8.98 ± 0.02 V; and gray circles, a = 0 ± 0 V, k[obs] = 0 ± 0 s^−1, b = 8.63 ± 0.01 V. These values are in excellent agreement with those reported previously in the absence of benzamidine (23). B, kinetic traces of Na^+ binding to the thrombin mutant N143P in the 0–250 ms time scale. Shown are the traces obtained at 0 (open circles) and 50 mm Na^+ in the absence (black circles) or presence (gray circles) of 1 mm benzamidine. Notice how the binding of Na^+ to N143P produces only a rapid phase of fluorescence enhancement comparable in total amplitude to that observed in the wild type. The presence of benzamidine reduces the amplitude of the fast phase and gives a total amplitude again comparable to that seen in the wild type. Experimental conditions are: 50 nm thrombin, 5 mm Tris, 0.1% PEG 8000, pH 8.0, at 15 °C. Continuous lines were drawn using the expression b − a exp(−k[obs]t) with best-fit parameter values: open circles, a = 0 ± 0 V, k[obs] = 0 ± 0 s^−1, b = 8.09 ± 0.01 V; black circles, a = 0 ± 0 V, k[obs] = 0 ± 0 s^−1, b = 8.87 ± 0.01 V; and gray circles, a = 0 ± 0 V, k[obs] = 0 ± 0 s^−1, b = 8.51 ± 0.01 V. C, Na^+ binding curves of thrombin wild-type (open circles) and N143P (black circles) obtained from the total change in intrinsic fluorescence determined by stopped-flow kinetics. Experimental conditions are: 50 nm thrombin, 5 mm Tris, 0.1% PEG 8000, pH 8.0, at 15 °C. The [Na^+] was changed by keeping the ionic strength constant at 400 mm with ChCl. Continuous lines were drawn according to the equation, F = (F[0] + F[1]K[app][Na^+])/(1 + K[app][Na^+]), where F[0] and F[1] are, respectively, the limiting values of intrinsic fluorescence in the absence and under saturating [Na^+] and K[app] = K[A]/(1 + r) is the apparent Na^+ binding affinity (23). The best-fit parameter values are: wild-type, F[0] = 8.07 ± 0.04 V, F[1] = 9.12 ± 0.03 V, K[app] = 120 ± 20 m^−1; N143P, F[0] = 8.10 ± 0.02 V, F[1] = 9.19 ± 0.02 V, K[app] = 52 ± 5 m^−1.
Figure 4.
X-ray crystal structure of the thrombin mutant N143P. Left: ribbon representation of the structure of the thrombin mutant N143P (Pro^143 is shown as a stick model and indicated by arrow) in the E* (cyan) and E:Na^+ (gold) forms. The 215–217 β-strand in the mutant collapses into the primary specificity pocket (red arrows), with the side chain of Trp^215 (stick model) repositioned into the active site (residues of the catalytic triad His^57, Asp^102, and Ser^195 are shown as stick models) in hydrophobic interaction with Trp^60d, Tyr^60a, Leu^99, and His^57. This represents a drastic change (r.m.s.d. 0.384 Å) from the E:Na^+ conformation of the mutant N143P (Na^+ shown as a purple ball) where the side chain of Trp^215 is positioned 10.5 Å away and leaves the active site accessible to substrate. Right: details of the disruption of the oxyanion hole in the thrombin mutant N143P in the E:Na^+ form (CPK, gold). The conformation of the same residues in the E* form is shown by comparison (CPK, cyan). The Glu^192-Gly^193 peptide bond is flipped (red arrow), because the presence of Pro^143 abolishes the H-bond between the backbone N atom of residue 143 and the backbone O atom of Glu^192. The 2F[o] − F[c] electron density map (light green mesh) refers to the E:Na^+ form and is contoured at 1.0 σ.
 
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 36175-36185) copyright 2009.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21406063 C.S.Craik, M.J.Page, and E.L.Madison (2011).
Proteases as therapeutics.
  Biochem J, 435, 1.  
21360607 H.C.Castro, P.A.Abreu, R.B.Geraldo, R.C.Martins, R.Dos Santos, N.I.Loureiro, L.M.Cabral, and C.R.Rodrigues (2011).
Looking at the proteases from a simple perspective.
  J Mol Recognit, 24, 165-181.  
21368156 S.Rana, N.Pozzi, L.A.Pelc, and E.Di Cera (2011).
Redesigning allosteric activation in an enzyme.
  Proc Natl Acad Sci U S A, 108, 5221-5225.  
20809655 A.D.Vogt, A.Bah, and E.Di Cera (2010).
Evidence of the E*-E equilibrium from rapid kinetics of Na+ binding to activated protein C and factor Xa.
  J Phys Chem B, 114, 16125-16130.  
20974933 Z.Chen, L.A.Pelc, and E.Di Cera (2010).
Crystal structure of prethrombin-1.
  Proc Natl Acad Sci U S A, 107, 19278-19283.
PDB code: 3nxp
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.