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Lucas Lu¨ thy, Markus G. Gru¨ tter, and Peer R. E. Mittl JOURNAL OF BIOLOGICAL CHEMISTRY

The Crystal Structure of Helicobacter pylori Cysteine-rich Protein B Reveals a Novel Fold for a Penicillin-binding Protein. Lucas Lu¨ thy, Markus G. Gru¨ tter, and Peer R. E. Mittl JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 12, Issue of March 22, pp. 10187–10193, 2002.

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Lucas Lu¨ thy, Markus G. Gru¨ tter, and Peer R. E. Mittl JOURNAL OF BIOLOGICAL CHEMISTRY

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  1. The Crystal Structure of Helicobacter pylori Cysteine-rich Protein BReveals a Novel Fold for a Penicillin-binding Protein Lucas Lu¨ thy, Markus G. Gru¨ tter, and Peer R. E. Mittl JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 12, Issue of March 22, pp. 10187–10193, 2002

  2. Introduction ofHelicobacter pylori • Colonization of the gastric mucosa with the spiral-shaped Gram-negative proteobacterium Helicobacter pylori is probably the most common chronic infection in humans. • gastric diseases • duodenal ulcers • non-gastric diseases • cardiovascular, autoimmune, dermatological, and liver diseases

  3. What isHelicobacter pylori Cysteine-rich Protein ? (Hcp) • In the H. pylori genomes ,two cysteine residues are separated by 7 amino acids, and there are 36 amino acids between adjacent cysteine pairs • Sequence conservation among this family varies between 22 and 66% sequence identity

  4. Multiple sequence alignment of the Hcp family

  5. The characteristic function of Hcp family • β -lactamase activity • penicillin binding activity

  6. β-Lactam Hydrolysis THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 23, Issue of June 9, pp. 17693–17699, 2000

  7. The Crystal Structure of HcpB

  8. Sample purification • Since HcpB was refolded from inclusion bodies, proper refolding was verified by CD spectroscopy.

  9. Characterization of Folding CD spectrum of refolded HcpB. Ellipticity at a wavelength of 222 nm as a function of GdmHCl concentration. Based on the CD spectrum, the –αhelix content was predicted to be 73%, [GdmHCl]1 /2 values is 1.93 +-0.02 M and free energy is unfolding of -22 kJ/mol.

  10. The 133 residues of HcpB fold into eight α-helices that pack into a right-handed superhelix • Four disulfide bridges are observed between cysteine pairs Cys-22/Cys-30, Cys-52/Cys-60, Cys-88/Cys-96, and Cys-124/Cys-132. • The disulfide bridges subdivide the structure into four (1, 2, 3, 4) pairs (A, B)of α-helices confirming the proposed modular architecture.

  11. The packing angle of helices A and B belonging to the same α/α –motif is 42° whereas the angle between helices B and A of adjacent motifs is 14° • The convex surface of the molecule is formed by helices 1A, 2A, 3A, and 4A. This surface area is positively charged. • Helices 1B, 2B, 3B, and 4B create an amphipathic groove. Polar side chains of helix 2B form the bottom of the grove that is flanked on both sides by hydrophobic side chains coming from helices 1B, 3B, and 4B.

  12. Sequence identity among α /α -motifs of HcpB

  13. R.m.s.d. and sequence identity among α / α -motifs of HcpB Stereo view of the superposition of four HcpB α / α -motifs.

  14. Structure-based sequence alignment of HcpB motifs 1–4 Residues that are conserved are highlighted The cysteine residues at positions 20 and 28, alanine at position 19, and glycine at position 27 are conserved for structural reasons. Throughout the whole Hcp family, residues preceding the cysteines are always glycine, alanine, or serine residues because these residue types possess sufficiently small side chains.

  15. The Binding Site

  16. Attempts to detect the nitrocefin binding site in HcpB failed because the crystals disintegrated upon soaking nitrocefin into the HcpB crystals • Mass spectrometric analysis of HcpB revealed two peaks with molecular masses of 16,159.2 and 16,450.8 Da , and the 292 Da difference between two peaks is suggesting the ligand in active site .

  17. N-acetylmuramic acid (NAM) is a compound that is found in the peptidoglycan of all Gram-negative bacteria.

  18. Water molecules and 2Fo-Fc electron density in the putative ligand binding site. Modeled N-acetylmuramic acid/HcpB complex..

  19. Comparability of the structure between HcpB and tetratricopeptide repeat (TPR) domain of the human protein phosphatase 5 (PP5)

  20. TPR motifs are important to the function of chaperone , cell-cycle transcription . BioEssays 21:932–939, r 1999 John Wiley & Sons, Inc.

  21. The structural differience between HcpB and PP5 The EMBO Journal Vol.17 No.5 pp.1192–1199, 1998

  22. Fig.2c Fig.2d leucine at position 22 is also conserved in TPR repeats 1 and 2, whereas the lysine and asparagine residues at positions 11, 14, and 18 that are located on the convex surface of HcpB are not.

  23. Conclusion • The 133 residues of HcpB fold into eight α-helices and four disulfide bridges are observed between each pair of the α/α –motif . • The ligand could form hydrogen bonds with residues in the loops between helices A and B of motifs 1, 2, and 3 • The structure of HcpB is most similar to the tetratricopeptide repeat (TPR) domain of the human protein phosphatase 5 (PP5) , but their function are very diverse .

  24. Thanks

  25. X-ray data collection and refinement statistics

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