Stefania Alleva 1 , Ruggero Tenni 2 , Anna Lupi 2 , Velia Minicozzi 1 , Silvia Morante 1 ,

1. 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 2Department of Biochemistry, University of Pavia - Pavia, Italy Acta Biophysica Romana 2008 10-11 Aprile 2008

2. 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

3. 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)Biochemistry43, 2771-2783

4. 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: HomoSapiens, Mouse, Pyrococcus Furiosus, E. Coli. … Previous evidences: Co+2 in PyrococcusF. and Mn+2 in Homo sapiens are needed for enzymatic activity. In both organisms Zn+2 suppresses enzymatic activity. Maher et al., (2004)Biochemistry43, 2771-2783

5. 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 Mn+2 [PrD]:[Mn+2]=1:4 Human Prolidase with Na+ [PrD]:[Na+]=1:5 “fifth site”? PDB ID 2okn PDB ID 2iw2 Maher et al. (2004)Biochemistry43, 2771-2783 Lupi et al., (2006) FEBS Journal 273, 5466-5478

6. 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

7. 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

8. 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 pH=7.8 Lupi et al., (2006) FEBS Journal 273, 5466-5478

9. 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 During the preparation, samples have been exposed to Zn+2 which replaces some of Mn+2 ions in the active site. Despite the presence of Zn+2 a high enzyme activityis registered Final Measurement [PrD] : [Zn+2] = 1:4 [PrD] : [Mn+2] = 1:1 Lupi et al., (2006) FEBS Journal 273, 5466-5478

10. 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 EXCURVE98package • 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

11. 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 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

12. The presence of a metal scatterer near the Mn+2 absorber is confirmed by EXAFS analysis Crystal structure Human Prolidase with Mn+2: differences among metal binding sites in the two monomers site B1 site C2 site A2 site A1 A1 = A2

13. PrD can exist in one of these structures a structure g structure b structure d structure

14. 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

15. 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 A1 Zn scatterer Zn absorber in site B1 Zn scatterer Zn absorber in site C2 Zn scatterer Zn absorber in site C2 Mn scatterer Zn absorber in site A1 Mn scatterer Zn absorber in site B1 Mn scatterer

16. (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 boundtoone 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