Diapositiva 1

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Identifying the structure of the active sites of
Human Prolidase
Stefania Alleva1, Ruggero Tenni2, Anna Lupi2,
Velia Minicozzi1, Silvia Morante1, Francesco
Stellato1, Antonella Forlino2 1 Department of
Physics, University of Rome Tor Vergata- Rome,
Italy 2 Department of Biochemistry, University of
Pavia - Pavia, Italy
Acta Biophysica Romana 2008 10-11 Aprile 2008
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• SUMMARY
• Prolidase protein structural and functional
  features
• Human Prolidase
• function and crystal structure
• Prolidase Deficiency
• XAS measurements on recombinant Human
  Prolidase
• and data analysis
• (Preliminary) Conclusion and work in progress

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Prolidase protein structural and functional
features Cytosolic Mn-dependent
exopeptidase Widespread in nature found in
different kinds of organisms (archea, bacteria
and eucarya) Dimeric protein To initiate the
dimerization process (and then protein
activation) metal ions are needed For the full
activation a di-nuclear metal site is
needed Functions It is involved in the final
state of metabolism of proline containing
proteins, thus cooperating in the proline
recycling Biotechnology use proline release
reduces foods bitterness
Maher et al., (2004) Biochemistry 43, 2771-2783
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The monomeric metal binding site is di-nuclear in
all studied organisms (h human)
High homology, in the active site regions, among
Prolidase sequences of different organisms Homo
Sapiens, Mouse, Pyrococcus Furiosus, E. Coli.
Previous evidences Co2 in Pyrococcus F. and
Mn2 in Homo sapiens are needed for enzymatic
activity. In both organisms Zn2 suppresses
enzymatic activity.
Maher et al., (2004) Biochemistry 43, 2771-2783
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• Human Prolidase
• function and crystal structure
• Homodimer each monomer composed of 492 a.a.
  (54.3 kDa)
• Hydrolysis of dipeptides X-Pro or X-Hyp at
  C-terminal

Known crystal structures of Human Prolidase
Human Prolidase with Mn2 PrDMn214
Human Prolidase with Na PrDNa15
fifth site?
PDB ID 2okn
PDB ID 2iw2
Maher et al. (2004) Biochemistry 43,
2771-2783 Lupi et al., (2006) FEBS Journal 273,
5466-5478
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Prolidase Deficency Reduced or depleted
Prolidase activity in humans cause Prolidase
Deficiency (PD) PD is a rare autosomic recessive
illness that affects about 1-2 every 106
people Clinical symptoms are skin
lesions mental retardation lung infection To
date no cure is known
Lupi et al. (2006) J. Med. Genet. 43, 58-63
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PD is caused by mutations in the Prolidase gene
(PEPD) located on chromosome 19
Molecular analysis on PD cases identifies 13
different mutations in PEPD For five of them
structural alterations - that modify protein
capability of binding metal ions with loss of
catalytic activity- have been reported
Arg184 ?Gln Gly278?Asp Glu412? Lys Asp276?
Asn Gly448? Arg
An important point is to understand the role
played by metal ions in the activation process
Lupi et al. (2006) J .Med. Genet. 43, 58-63
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184
Arg
Gln
278
Gly
Asp
in PD patients
a.a. substitution
412
Lys
Glu
276
Asp
Asn
448
Gly
Arg
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XAS measurements and data analysis on
recombinant Human Prolidase Recombinant Human
Prolidase is generated in eukaryotic (CHO) and
prokaryotic (E.Coli) hosts (Department of
Biochemistry - University of Pavia) Recombinant
Prolidase has the same biochemical properties as
the endogenous Human enzyme (substrate
specificity, optimal temperature and pH, metal
dependence)
Metal ion dependence
Substrate specificity
Dimeric recombinant protein (PrD) from E.Coli
purified through imidazole step gradient and
suspended in 10 mM Tris-HCl, 0.57 mM DTT, 0.3 M
NaCl at pH7.8
Lupi et al., (2006) FEBS Journal 273, 5466-5478
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Two samples at different PrD concentration
XAS1 PrD 0.03 mM XAS2 PrD 0.35
mM GSH MnCl2
ICP-MS relieves some metals in trace and
expecially Zn2
During the preparation, samples have been exposed
to Zn2 which replaces some of Mn2 ions in the
active site.
Despite the presence of Zn2 a high enzyme
activity is registered
Final Measurement
PrD Zn2 14 PrD Mn2 11
Lupi et al., (2006) FEBS Journal 273, 5466-5478
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Spectra have been collected at Mn and Zn K-edge
from both samples XAS1 and XAS2 and also from Mn
and Zn in buffer.
DATA ANALYSIS
EXCURVE98 package

• separate inter- and intra-ligand multiple
  scattering paths
• treat chemical groups like rigid units
• use PDB format for input and output file

Beamline D2 EMBL Desy outstation, Hamburg
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Estimate of PrD concentration (Lowry assay) and
metal concentration (EXAFS non normalized
spectra) PrDZn2 14 PrDMn2
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Prolidase has 5 binding sites for metals four
occupied by Zn and one by Mn
Identify Mn binding site
Hypothetical FIFTH SITE
Assuming Mn in the fifth site Scatterers 3 O
Known X-ray cristallography
Fit seems to exclude Mn ? Zn
Mn
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The presence of a metal scatterer near the Mn2
absorber is confirmed by EXAFS analysis
Crystal structure Human Prolidase with
Mn2 differences among metal binding sites in
the two monomers
site B1
site C2
site A2
site A1
A1 A2
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PrD can exist in one of these structures
a structure
g structure
b structure
d structure
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• Mn absorber in site A1, B1 or C2
• Zn scatterer

Spectra at the Mn edge
Mn absorber in site C2 Scatterers 6 O 1 Zn
Mn absorber in site A1 Scatterers 4 O 1 His 1
Zn
Mn absorber in site B1 Scatterers 5 O 1 Zn
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Spectra at the Zn edge

• More complicated situation
• four Zn ions per dimer
• Zn in the fifth site
• Mn or Zn as metal scatterer

Zn absorber in site B1 Zn scatterer
Zn absorber in site C2 Zn scatterer
Zn absorber in site A1 Zn scatterer
Zn absorber in site C2 Mn scatterer
Zn absorber in site B1 Mn scatterer
Zn absorber in site A1 Mn scatterer
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(Preliminary) Conclusions Full enzymatic activity
in the presence of bound Zn 5 metal binding sites
in each PrD Zn2 bound to the fifth site
Mn2 bound to one of the two di-nuclear sites
(possibly B1 or C2) Work in progress Identificati
on of the Mn binding site structure Analysis of
the multiple site geometry occurring for Zn ions
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