Stefania Alleva1, Ruggero Tenni2, Anna Lupi2, Velia Minicozzi1, Silvia Morante1,Francesco Stellato1, Antonella Forlino2

1 Department of Physics, University of Rome “Tor Vergata”- Rome, Italy2 Department of Biochemistry, University of Pavia - Pavia, Italy

Acta Biophysica Romana 2008 10-11 Aprile 2008

Identifying the structure of the active sites of Human Prolidase

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

Prolidase protein:structural and functional features

Cytosolic Mn-dependent exopeptidase

Widespread in nature: found in different kinds of organisms(archea, bacteria and eucarya)

Dimeric proteinTo initiate the dimerization process (and then protein activation)

metal ions are neededFor 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

High homology, in the active site regions, among Prolidase sequences of

different organisms:Homo Sapiens, Mouse, Pyrococcus Furiosus, E. Coli. …

The monomeric metal binding site is di-nuclear in

all studied organisms

(h = human)

Previous evidences: Co+2 in Pyrococcus F. and Mn+2 in Homo sapiens are needed for enzymatic

activity. In both organisms Zn+2 suppresses enzymatic activity.

Maher et al., (2004) Biochemistry 43, 2771-2783

Human Prolidasefunction 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

Maher et al. (2004) Biochemistry 43, 2771-2783

Lupi et al., (2006) FEBS Journal 273, 5466-5478

Human Prolidase with Mn+2

[PrD]:[Mn+2]=1:4

Human Prolidase with Na+

[PrD]:[Na+]=1:5

PDB ID 2okn PDB ID 2iw2

Known crystal structures of Human Prolidase

“fifth site”?

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 areskin lesionsmental retardationlung infection

To date no cure is known

Lupi et al. (2006) J. Med. Genet. 43, 58-63

Lupi et al. (2006) J .Med. Genet. 43, 58-63

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

PD is caused by mutations in the Prolidase gene (PEPD) located on chromosome 19

An important point is to understand

the role played by metal ions in the

activation process

Arg184 →Gln

Gly278→Asp

Glu412→ Lys

Asp276→ Asn

Gly448→ Arg

XAS measurements and data analysis on

recombinant Human ProlidaseRecombinant 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)

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 pH=7.8

Metal ion dependence Substrate specificity

Lupi et al., (2006) FEBS Journal 273, 5466-5478

During the preparation, samples have been exposed to Zn+2 which replaces some of

Mn+2 ions in the active site.

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 Zn+2

Despite the presence of Zn+2

a high enzyme activity is registered

Lupi et al., (2006) FEBS Journal 273, 5466-5478

[PrD] : [Zn+2] = 1:4[PrD] : [Mn+2] = 1:1

Final Measurement

Spectra have been collected at Mn and Zn K-edge from both samples XAS1 and XAS2 and also from Mn and Zn in buffer.

Beamline D2 EMBL

Desy outstation, Hamburg

EXCURVE98 package

• separate inter- and intra-ligand multiple scattering paths

• treat chemical groups like rigid units

• use PDB format for input and output file

DATA ANALYSIS

Hypothetical “FIFTH SITE”

Assuming Mn in the “fifth site”

Scatterers: 3 O

Mn

Estimate of PrD concentration (Lowry assay) and metal concentration (EXAFS non normalized spectra)

[PrD]:[Zn+2] = 1:4[PrD]:[Mn+2] = 1:1

Prolidase has 5 binding sites for metals:four occupied by Zn and one by Mn

Known X-ray cristallography

Fit seems to exclude Mn

↓ Zn

Identify Mn

binding site

Crystal structure Human Prolidase with Mn+2:differences among metal binding sites in the two monomers

Monomer 1 Monomer 2

site A1 site B1 site A2 site C2

4 O 5 O 4 O 6 O

1 His 1 His

1 Mn 1 Mn 1 Mn 1 Mn

site A1site A2

site B1 site C2

A1 = A2

The presence of a metal scatterer

near the Mn+2 absorber is confirmed by EXAFS analysis

Structure name Monomer 1 Monomer 2

site A1 site B1 site A2 site C2

Zn Mn Zn Zn

Mn Zn Zn Zn

Zn Zn Mn Zn

Zn Zn Zn Mn

PrD can exist in one of these structures

structure

structure structure

structure

Mn absorber in site B1Scatterers: 5 O + 1 Zn

Mn absorber in site A1Scatterers: 4 O + 1 His+ 1 Zn

Spectra at the Mn edge Mn absorber in site A1, B1 or C2

Zn scatterer

Mn absorber in site C2Scatterers: 6 O + 1 Zn

Spectra at the Zn edgeMore complicated situation

four Zn ions per dimer Zn in the “fifth site” Mn or Zn as metal scatterer

Zn absorber in site B1Zn scatterer

Zn absorber in site B1Mn scatterer

Zn absorber in site C2Zn scatterer

Zn absorber in site C2Mn scatterer

Zn absorber in site A1Zn scatterer

Zn absorber in site A1Mn scatterer

(Preliminary) Conclusions

Full enzymatic activity in the presence of bound Zn

5 metal binding sites in each PrD:

Zn+2 bound to the “fifth site”

Mn+2 bound to one of the two di-nuclear sites

(possibly B1 or C2)

Work in progress

Identification of the Mn binding site structure

Analysis of the multiple site geometry occurring for Zn ions