鉤端螺旋體病是溫熱帶國家常見的人畜共通疾病之一，其致病的病原菌為鉤端螺旋體。此疾病可能導致多重器官衰竭，而腎臟是感染最好發之處。目前對於鉤端螺旋體如何引起宿主的先天免疫反應，以及感染腎臟的生理影響仍不清楚。鉤端螺旋體病相關致病因子多數為外膜蛋白，外膜蛋白位於病原體的表面，被免疫系統辨識而誘發免疫反應的機率較其他蛋白高，故常扮演毒素因子的角色，也經常被用來作為疫苗研發之標的；除此之外，外膜蛋白在病原體與宿主間交互作用中伴演著重要的角色。LipL41為鉤端螺旋體中高度保留且是主要的外膜脂質蛋白之一。先前的研究發現LipL41具有高鐵血紅素（hemin）的結合能力，且有可能參與病原體的鐵離子調控與儲存。然而，LipL41與高鐵血紅素結合的詳細機制尚未被明確地探討。在本研究中，我們透過高鐵血紅素結合分析法求得了LipL41和高鐵血紅素之間的解離常數（Dissociation constant, Kd）為0.59 ± 0.14 μM；同時，我們也鑑定出位於LipL41之上的可能參與高鐵血紅素結合的結合位（HRMs, heme regulatory motif）。藉由突變重組蛋白LipL41，我們也證實了LipL41上的兩個胺基酸Cys140以及Cys220同時參與在與高鐵血紅素的結合。我們也運用了穿透式電子顯微鏡發現了LipL41會形成一具有36個單體的超分子聚合體。此外，藉由圓二色光譜及核磁共振的分析，發現LipL41碳端包含兩個TPR motif的區域（LipL41-C100）具有熔球態蛋白（molten globule protein）的特性。

Leptospirosis is one of the most widespread zoonotic diseases in the world. It is caused by the pathogen Leptospira that results in multiple-organ failure, in particular of the kidney. Outer membrane lipoprotein is the suspected virulence factor of Leptospira. In Leptospira spp LipL41 is one major lipoprotein and is highly conserved. Previous study suggests that LipL41 bears hemin-binding ability and might play a possible role in iron regulation and storage. However, the characterization of hemin-binding ability of LipL41 is still unclear. Here the hemin-binding ability of LipL41 was examined, yielding a Kd = 0.59 ± 0.14 μM. Two possible heme regulatory motifs (HRMs), C[P/S], were found in LipL41 at 140Cys-Ser and 220Cys-Pro. The mutational study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. The LipL41 oligomer consists of 36 molecules and folds as a double-layered yo-yo particle. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might be involved in the protein-protein interaction of the supramolecular assembly. Besides, the TPR-containing fragment, LipL41-C100, shows thermal stability and reversibility by circular dichroism and NMR. The flexible folding of LipL41-C100 can be stabilized by adding 50% TFE. The thermal dynamics and flexible folding consider that LipL41-C100 is a molten globule protein, which conserves a native-like secondary structure content but without the tightly packed protein interior.

1. Levett PN (2001) Leptospirosis. Clin Microbiol Rev 14: 296-326.
2. Farr RW (1995) Leptospirosis. Clin Infect Dis 21: 1-6.
3. Yang CW (2007) Leptospirosis renal disease: Understanding the initiation by Toll-like receptors. Kidney Int 72: 918-925.
4. Dolhnikoff M, Mauad T, Bethlem EP, Carvalho CR (2007) Pathology and pathophysiology of pulmonary manifestations in leptospirosis. Braz J Infect Dis 11: 142-148.
5. Yang CW, Wu MS, Pan MJ (2001) Leptospirosis renal disease. Nephrol Dial Transplant 16 Suppl 5: 73-77.
6. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, et al. (2003) Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3: 757-771.
7. Tian YC, Chen YC, Hung CC, Chang CT, Wu MS, et al. (2006) Leptospiral outer membrane protein induces extracellular matrix accumulation through a TGF-beta1/Smad-dependent pathway. J Am Soc Nephrol 17: 2792-2798.
8. Vinh T, Adler B, Faine S (1986) Ultrastructure and chemical composition of lipopolysaccharide extracted from Leptospira interrogans serovar Copenhageni. J Gen Microbiol 132: 103-109.
9. Haake DA, Chao G, Zuerner RL, Barnett JK, Barnett D, et al. (2000) The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection. Infect Immun 68: 2276-2285.
10. Lo YY, Hsu SH, Ko YC, Hung CC, Chang MY, et al. (2013) Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway. J Biol Chem 288: 12335-12344.
11. Barnett JK, Barnett D, Bolin CA, Summers TA, Wagar EA, et al. (1999) Expression and distribution of leptospiral outer membrane components during renal infection of hamsters. Infect Immun 67: 853-861.
12. Tung JY, Yang CW, Chou SW, Lin CC, Sun YJ (2010) Calcium binds to LipL32, a lipoprotein from pathogenic Leptospira, and modulates fibronectin binding. J Biol Chem 285: 3245-3252.
13. Yang CW, Wu MS, Pan MJ, Hsieh WJ, Vandewalle A, et al. (2002) The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells. J Am Soc Nephrol 13: 2037-2045.
14. Yang CW, Hung CC, Wu MS, Tian YC, Chang CT, et al. (2006) Toll-like receptor 2 mediates early inflammation by leptospiral outer membrane proteins in proximal tubule cells. Kidney Int 69: 815-822.
15. Yang CW, Wu MS, Pan MJ, Hong JJ, Yu CC, et al. (2000) Leptospira outer membrane protein activates NF-κB and downstream genes expressed in medullary thick ascending limb cells. J Am Soc Nephrol 11: 2017-2026.
16. Ko AI, Goarant C, Picardeau M (2009) Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nat Rev Microbiol 7: 736-747.
17. Sutcliffe IC, Russell RR (1995) Lipoproteins of gram-positive bacteria. J Bacteriol 177: 1123-1128.
18. Hayashi S, Wu HC (1990) Lipoproteins in bacteria. J Bioenerg Biomembr 22: 451-471.
19. Sankaran K, Wu HC (1994) Lipid modification of bacterial prolipoprotein. Transfer of diacylglyceryl moiety from phosphatidylglycerol. J Biol Chem 269: 19701-19706.
20. Hantke K, Braun V (1973) Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur J Biochem 34: 284-296.
21. Sritharan M (2000) Iron as a candidate in virulence and pathogenesis in mycobacteria and other microorganisms. World J Microbiol Biotechnol 16: 769-780.
22. Murray GL, Srikram A, Henry R, Puapairoj A, Sermswan RW, et al. (2009) Leptospira interrogans requires heme oxygenase for disease pathogenesis. Microbes Infect 11: 311-314.
23. Griffiths E (1999) Iron in biological systems. In: Bullen JJ, Griffiths E, editors. Iron and infection: molecular, physiological and clinical aspects. New York, NY: John Wiley & Sons. pp. 1-26.
24. Caughey WS, Smythe GA, O'Keeffe DH, Maskasky JE, Smith MI (1975) Heme A of cytochrome c oxicase. Structure and properties: comparisons with hemes B, C, and S and derivatives. J Biol Chem 250: 7602-7622.
25. Henderson DP, Payne SM (1994) Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins. J Bacteriol 176: 3269-3277.
26. Torres AG, Payne SM (1997) Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7. Mol Microbiol 23: 825-833.
27. Mills M, Payne SM (1997) Identification of shuA, the gene encoding the heme receptor of Shigella dysenteriae, and analysis of invasion and intracellular multiplication of a shuA mutant. Infect Immun 65: 5358-5363.
28. Binet R, Wandersman C (1996) Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: A TolC analogue. Mol Microbiol 22: 265-273.
29. Letoffe S, Redeker V, Wandersman C (1998) Isolation and characterization of an extracellular haem-binding protein from Pseudomonas aeruginosa that shares function and sequence similarities with the Serratia marcescens HasA haemophore. Mol Microbiol 28: 1223-1234.
30. Braun V, Braun M (2002) Active transport of iron and siderophore antibiotics. Curr Opin Microbiol 5: 194-201.
31. Haake DA, Walker EM, Blanco DR, Bolin CA, Miller MN, et al. (1991) Changes in the surface of Leptospira interrogans serovar Grippotyphosa during in vitro cultivation. Infect Immun 59: 1131-1140.
32. Shang ES, Summers TA, Haake DA (1996) Molecular cloning and sequence analysis of the gene encoding LipL41, a surface-exposed lipoprotein of pathogenic Leptospira species. Infect Immun 64: 2322-2330.
33. Natarajaseenivasan K, Vijayachari P, Sharma S, Sugunan AP, Selvin J, et al. (2008) Serodiagnosis of severe leptospirosis: evaluation of ELISA based on the recombinant OmpL1 or LipL41 antigens of Leptospira interrogans serovar Autumnalis. Ann Trop Med Parasitol 102: 699-708.
34. Mariya R, Chaudhary P, Kumar AA, Thangapandian E, Amutha R, et al. (2006) Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar Canicola in ELISA for serodiagnosis of bovine leptospirosis. Comp Immunol Microbiol Infect Dis 29: 269-277.
35. King AM, Bartpho T, Sermswan RW, Bulach DM, Eshghi A, et al. (2013) Leptospiral outer membrane protein LipL41 is not essential for acute leptospirosis but requires a small chaperone protein, lep, for stable expression. Infect Immun 81: 2768-2776.
36. Asuthkar S, Velineni S, Stadlmann J, Altmann F, Sritharan M (2007) Expression and characterization of an iron-regulated hemin-binding protein, HbpA, from Leptospira interrogans serovar Lai. Infect Immun 75: 4582-4591.
37. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
38. Davis BJ (1964) Disc Electrophoresis. Ii. Method and Application to Human Serum Proteins. Ann N Y Acad Sci 121: 404-427.
39. Schuck P, Perugini MA, Gonzales NR, Howlett GJ, Schubert D (2002) Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. Biophys J 82: 1096-1111.
40. Barltrop JA, Owen TC, Cory AH, Cory JG (1991) 5-(3-Carboxymethoxyphenyl)-2-(4,5-Dimethylthiazolyl)-3-(4-Sulfophenyl)Tetrazolium, Inner Salt (Mts) and Related Analogs of 3-(4,5-Dimethylthiazolyl)-2,5-Diphenyltetrazolium Bromide (Mtt) Reducing to Purple Water-Soluble Formazans as Cell-Viability Indicators. Bioorganic & Medicinal Chemistry Letters 1: 611-614.
41. Lee BC (1992) Isolation of an outer membrane hemin-binding protein of Haemophilus influenzae type b. Infect Immun 60: 810-816.
42. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9: 671-675.
43. Valcu M, Valcu CM (2011) Data transformation practices in biomedical sciences. Nat Methods 8: 104-105.
44. Bohm G, Muhr R, Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 5: 191-195.
45. Tang G, Peng L, Baldwin PR, Mann DS, Jiang W, et al. (2007) EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol 157: 38-46.
46. Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, et al. (2013) CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res 41: D348-352.
47. Biegert A, Mayer C, Remmert M, Soding J, Lupas AN (2006) The MPI Bioinformatics Toolkit for protein sequence analysis. Nucleic Acids Res 34: W335-339.
48. D'Andrea LD, Regan L (2003) TPR proteins: the versatile helix. Trends Biochem Sci 28: 655-662.
49. Zeytuni N, Zarivach R (2012) Structural and functional discussion of the tetra-trico-peptide repeat, a protein interaction module. Structure 20: 397-405.
50. Oldfield CJ, Cheng Y, Cortese MS, Brown CJ, Uversky VN, et al. (2005) Comparing and combining predictors of mostly disordered proteins. Biochemistry 44: 1989-2000.
51. Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O, et al. (2005) FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21: 3435-3438.
52. Lathrop JT, Timko MP (1993) Regulation by heme of mitochondrial protein-transport through a conserved amino-acid motif. Science 259: 522-525.
53. Zhang L, Guarente L (1995) Heme binds to a short sequence that serves a regulatory function in diverse proteins. EMBO J 14: 313-320.
54. Qi ZH, Hamza I, O'Brian MR (1999) Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Proc Natl Acad Sci U S A 96: 13056-13061.
55. Huang TJ, McCoubrey WK, Jr., Maines MD (2001) Heme oxygenase-2 interaction with metalloporphyrins: function of heme regulatory motifs. Antioxid Redox Signal 3: 685-696.
56. Yang F, Xia X, Lei HY, Wang ED (2010) Hemin binds to human cytoplasmic arginyl-tRNA synthetase and inhibits its catalytic activity. J Biol Chem 285: 39437-39446.
57. Westberg JA, Jiang J, Andersson LC (2011) Stanniocalcin 1 binds hemin through a partially conserved heme regulatory motif. Biochem Biophys Res Commun 409: 266-269.
58. Kuhl T, Sahoo N, Nikolajski M, Schlott B, Heinemann SH, et al. (2011) Determination of hemin-binding characteristics of proteins by a combinatorial peptide library approach. Chembiochem 12: 2846-2855.
59. Hsu SH, Lo YY, Tung JY, Ko YC, Sun YJ, et al. (2010) Leptospiral outer membrane lipoprotein LipL32 binding on toll-like receptor 2 of renal cells as determined with an atomic force microscope. Biochemistry 49: 5408-5417.
60. Yi L, Morgan JT, Ragsdale SW (2010) Identification of a thiol/disulfide redox switch in the human BK channel that controls its affinity for heme and CO. J Biol Chem 285: 20117-20127.
61. Lin HH, Tseng LY (2010) DBCP: a web server for disulfide bonding connectivity pattern prediction without the prior knowledge of the bonding state of cysteines. Nucleic Acids Res 38: W503-W507.
62. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, et al. (2004) UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25: 1605-1612.
63. Fischer H, Polikarpov I, Craievich AF (2004) Average protein density is a molecular-weight-dependent function. Protein Science 13: 2825-2828.
64. Gautier R, Douguet D, Antonny B, Drin G (2008) HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 24: 2101-2102.
65. Brändén C-I, Tooze J (2009) Introduction to protein structure. New York, NY: Garland Pub.
66. Kazakov AS, Markov DI, Gusev NB, Levitsky DI (2009) Thermally induced structural changes of intrinsically disordered small heat shock protein Hsp22. Biophys Chem 145: 79-85.
67. Shiraki K, Nishikawa K, Goto Y (1995) Trifluoroethanol-induced stabilization of the alpha-helical structure of beta-lactoglobulin: implication for non-hierarchical protein folding. J Mol Biol 245: 180-194.
68. Fioroni M, Diaz MD, Burger K, Berger S (2002) Solvation phenomena of a tetrapeptide in water/trifluoroethanol and water/ethanol mixtures: a diffusion NMR, intermolecular NOE, and molecular dynamics study. J Am Chem Soc 124: 7737-7744.
69. Diaz MD, Fioroni M, Burger K, Berger S (2002) Evidence of complete hydrophobic coating of bombesin by trifluoroethanol in aqueous solution: an NMR spectroscopic and molecular dynamics study. Chemistry 8: 1663-1669.
70. Reiersen H, Rees AR (2000) Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains. Protein Eng 13: 739-743.
71. Tompa P (2002) Intrinsically unstructured proteins. Trends Biochem Sci 27: 527-533.
72. Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6: 197-208.
73. Dunker AK, Silman I, Uversky VN, Sussman JL (2008) Function and structure of inherently disordered proteins. Curr Opin Struct Biol 18: 756-764.
74. Faine S (1959) Iron as a growth requirement for pathogenic Leptospira. J Gen Microbiol 20: 246-251.
75. Lo M, Murray GL, Khoo CA, Haake DA, Zuerner RL, et al. (2010) Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog. Infect Immun 78: 4850-4859.
76. Cullen PA, Cordwell SJ, Bulach DM, Haake DA, Adler B (2002) Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect Immun 70: 2311-2318.
77. Picardeau M, Bulach DM, Bouchier C, Zuerner RL, Zidane N, et al. (2008) Genome sequence of the saprophyte Leptospira biflexa provides insights into the evolution of Leptospira and the pathogenesis of leptospirosis. PLoS One 3: e1607.
78. Cullen PA, Haake DA, Adler B (2004) Outer membrane proteins of pathogenic spirochetes. FEMS Microbiol Rev 28: 291-318.
79. Lewin A, Moore GR, Le Brun NE (2005) Formation of protein-coated iron minerals. Dalton Trans: 3597-3610.
80. Frolow F, Kalb AJ, Yariv J (1994) Structure of a unique twofold symmetric haem-binding site. Nat Struct Biol 1: 453-460.
81. Cliff MJ, Williams MA, Brooke-Smith J, Barford D, Ladbury JE (2005) Molecular recognition via coupled folding and binding in a TPR domain. J Mol Biol 346: 717-732.