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Fibronectin Binds to and Induces Conformational Change in a Disordered Region of Leptospiral Immunoglobulin-like Protein B

2009; Elsevier BV; Volume: 284; Issue: 35 Linguagem: Inglês

10.1074/jbc.m109.031369

ISSN

1083-351X

Autores

Yi‐Pin Lin, Alex D. Greenwood, Linda K. Nicholson, Yogendra Sharma, Sean P. McDonough, Yung‐Fu Chang,

Tópico(s)

Veterinary medicine and infectious diseases

Resumo

Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (KD = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (Kav) measured with gel permeation chromatography, a high hydrodynamic radius (Rh) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional 15N-1H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a β-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a β-strand-rich structure, suggestive of the known β-zipper mode of NTD binding. Leptospira interrogans is a pathogenic spirochete that causes disease in both humans and animals. LigB (Leptospiral immunoglobulin-like protein B) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin (Fn), fibrinogen, laminin, and collagen. A high affinity Fn-binding region of LigB has been recently localized to LigBCen2, which contains the partial eleventh and full twelfth immunoglobulin-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, LigBCen2NR was shown to bind to the N-terminal domain (NTD) of Fn (KD = 379 nm) by an enzyme-linked immunosorbent assay and isothermal titration calorimetry. Interestingly, this sequence was not observed to adopt secondary structure by far UV circular dichroism or by differential scanning calorimetry, in agreement with computer-based secondary structure predictions. A low partition coefficient (Kav) measured with gel permeation chromatography, a high hydrodynamic radius (Rh) measured with dynamic light scattering, and the insensitivity of the intrinsic viscosity to guanidine hydrochloride treatment all suggest that LigBCen2NR possesses an extended and disordered structure. Two-dimensional 15N-1H HSQC NMR spectra of intact LigBCen2 in the absence and presence of NTD are consistent with these observations, suggesting the presence of both a β-rich region and an unstructured region in LigBCen2 and that the latter of these selectively interacts with NTD. Upon binding to NTD, LigBCen2NR was observed by CD to adopt a β-strand-rich structure, suggestive of the known β-zipper mode of NTD binding. Leptospira interrogans is a pathogenic spirochete that causes leptospirosis throughout the world, especially in developing countries but also in regions of the United States where it has reemerged (1.Faine S.B. Adher B. Bolin C. Perolat P. Leptospira and Leptospirosis. MedSci, Medbourne, Australia1999: 67-69Google Scholar). Weil's syndrome, a severe form of this disease, is an acute febrile illness associated with multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, and meningitis (1.Faine S.B. Adher B. Bolin C. Perolat P. Leptospira and Leptospirosis. MedSci, Medbourne, Australia1999: 67-69Google Scholar), and has a 15% mortality rate if not treated (2.Segura E.R. Ganoza C.A. Campos K. Ricaldi J.N. Torres S. Silva H. Céspedes M.J. Matthias M.A. Swancutt M.A. López Liñán R. Gotuzzo E. Guerra H. Gilman R.H. Vinetz J.M. Clin. Infect. Dis. 2005; 40: 343-351Crossref PubMed Scopus (163) Google Scholar). The molecular pathogenesis of leptospirosis is poorly understood, and the bacterial virulence factors involved are largely unknown. Recently, several potential Leptospira virulence factors have been described, including sphingomyelinases, serine proteases, zinc-dependent proteases, and collagenase (3.Bulach D.M. Zuerner R.L. Wilson P. Seemann T. McGrath A. Cullen P.A. Davis J. Johnson M. Kuczek E. Alt D.P. Peterson-Burch B. Coppel R.L. Rood J.I. Davies J.K. Adler B. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 14560-14565Crossref PubMed Scopus (271) Google Scholar); LipL32 (4.Yang C.W. Wu M.S. Pan M.J. Hsieh W.J. Vandewalle A. Huang C.C. J. Am. Soc. Nephrol. 2002; 13: 2037-2045Crossref PubMed Scopus (100) Google Scholar); lipopolysaccharide (5.Werts C. Tapping R.I. Mathison J.C. Chuang T.H. Kravchenko V. Saint Girons I. Haake D.A. Godowski P.J. Hayashi F. Ozinsky A. Underhill D.M. Kirschning C.J. Wagner H. Aderem A. Tobias P.S. Ulevitch R.J. Nat. Immunol. 2001; 2: 346-352Crossref PubMed Scopus (573) Google Scholar); a novel factor H, laminin, and Fn-binding protein (Lsa24 or Len) (6.Barbosa A.S. Abreu P.A. Neves F.O. Atzingen M.V. Watanabe M.M. Vieira M.L. Morais Z.M. Vasconcellos S.A. Nascimento A.L. Infect. Immun. 2006; 74: 6356-6364Crossref PubMed Scopus (151) Google Scholar, 7.Stevenson B. Choy H.A. Pinne M. Rotondi M.L. Miller M.C. Demoll E. Kraiczy P. Cooley A.E. Creamer T.P. Suchard M.A. Brissette C.A. Verma A. Haake D.A. PLoS ONE. 2007; 2: e1188Crossref PubMed Scopus (163) Google Scholar, 8.Verma A. Hellwage J. Artiushin S. Zipfel P.F. Kraiczy P. Timoney J.F. Stevenson B. Infect. Immun. 2006; 74: 2659-2666Crossref PubMed Scopus (124) Google Scholar); Loa 22 (9.Ristow P. Bourhy P. da Cruz McBride F.W. Figueira C.P. Huerre M. Ave P. Girons I.S. Ko A.I. Picardeau M. PLoS Pathog. 2007; 3: e97Crossref PubMed Scopus (181) Google Scholar); and Lig (Leptospiral immunoglobulin-like) proteins (10.Matsunaga J. Barocchi M.A. Croda J. Young T.A. Sanchez Y. Siqueira I. Bolin C.A. Reis M.G. Riley L.W. Haake D.A. Ko A.I. Mol. Microbiol. 2003; 49: 929-945Crossref PubMed Scopus (214) Google Scholar, 11.Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar, 12.Palaniappan R.U. Chang Y.F. Jusuf S.S. Artiushin S. Timoney J.F. McDonough S.P. Barr S.C. Divers T.J. Simpson K.W. McDonough P.L. Mohammed H.O. Infect. Immun. 2002; 70: 5924-5930Crossref PubMed Scopus (125) Google Scholar). Lig proteins, including LigA, LigB, and LigC, contain multiple immunoglobulin-like repeat domains (13 in LigA, 12 in LigB and LigC) (10.Matsunaga J. Barocchi M.A. Croda J. Young T.A. Sanchez Y. Siqueira I. Bolin C.A. Reis M.G. Riley L.W. Haake D.A. Ko A.I. Mol. Microbiol. 2003; 49: 929-945Crossref PubMed Scopus (214) Google Scholar, 11.Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar, 12.Palaniappan R.U. Chang Y.F. Jusuf S.S. Artiushin S. Timoney J.F. McDonough S.P. Barr S.C. Divers T.J. Simpson K.W. McDonough P.L. Mohammed H.O. Infect. Immun. 2002; 70: 5924-5930Crossref PubMed Scopus (125) Google Scholar). Interestingly, the first 630 residues, from the N terminus to the first half of the seventh immunoglobulin-like domain, are conserved between LigA and LigB, but the rest of the immunoglobulin-like domains are variable (10.Matsunaga J. Barocchi M.A. Croda J. Young T.A. Sanchez Y. Siqueira I. Bolin C.A. Reis M.G. Riley L.W. Haake D.A. Ko A.I. Mol. Microbiol. 2003; 49: 929-945Crossref PubMed Scopus (214) Google Scholar, 11.Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar, 12.Palaniappan R.U. Chang Y.F. Jusuf S.S. Artiushin S. Timoney J.F. McDonough S.P. Barr S.C. Divers T.J. Simpson K.W. McDonough P.L. Mohammed H.O. Infect. Immun. 2002; 70: 5924-5930Crossref PubMed Scopus (125) Google Scholar) between the two proteins. Also, a non-immunoglobulin-like repeat region found on the C-terminal tail of LigB is not found in LigA (10.Matsunaga J. Barocchi M.A. Croda J. Young T.A. Sanchez Y. Siqueira I. Bolin C.A. Reis M.G. Riley L.W. Haake D.A. Ko A.I. Mol. Microbiol. 2003; 49: 929-945Crossref PubMed Scopus (214) Google Scholar, 11.Palaniappan R.U. Chang Y.F. Hassan F. McDonough S.P. Pough M. Barr S.C. Simpson K.W. Mohammed H.O. Shin S. McDonough P. Zuerner R.L. Qu J. Roe B. J. Med. Microbiol. 2004; 53: 975-984Crossref PubMed Scopus (69) Google Scholar, 12.Palaniappan R.U. Chang Y.F. Jusuf S.S. Artiushin S. Timoney J.F. McDonough S.P. Barr S.C. Divers T.J. Simpson K.W. McDonough P.L. Mohammed H.O. Infect. Immun. 2002; 70: 5924-5930Crossref PubMed Scopus (125) Google Scholar). Lig proteins are categorized as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) 2The abbreviations used are: MSCRAMMmicrobial surface component recognizing adhesive matrix moleculesDLSdynamic light scatteringDSCdifferential scanning calorimetryFnfibronectinGBDgelatin binding domainITCisothermal titration calorimetryMALDI-TOFmatrix-assisted laser desorption ionization-time of flightNTDN-terminal domainSPRsurface plasmon resonanceGSTglutathione S-transferaseELISAenzyme-linked immunosorbent assay.2The abbreviations used are: MSCRAMMmicrobial surface component recognizing adhesive matrix moleculesDLSdynamic light scatteringDSCdifferential scanning calorimetryFnfibronectinGBDgelatin binding domainITCisothermal titration calorimetryMALDI-TOFmatrix-assisted laser desorption ionization-time of flightNTDN-terminal domainSPRsurface plasmon resonanceGSTglutathione S-transferaseELISAenzyme-linked immunosorbent assay. due to their ability to bind to eukaryotic cells (13.Lin Y.P. Chang Y.F. J. Vet. Sci. 2008; 9: 133-144Crossref PubMed Scopus (42) Google Scholar) through their interactions with extracellular matrix components, including fibronectin (Fn), laminin, collagens, elastin, and tropoelastin (13.Lin Y.P. Chang Y.F. J. Vet. Sci. 2008; 9: 133-144Crossref PubMed Scopus (42) Google Scholar, 14.Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar, 45.Lin Y.P. Lee D.W. McDonough S.P. Nicholson L.K. Sharma Y. Chang Y.F. J. Biol. Chem. 2009; 284: 19380-19391Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Previously, a high affinity Fn-binding region was localized to LigBCen2, which includes the partial eleventh and complete twelfth immunoglobulin-like repeat region and the first 47 amino acids of the non-repeat regions of LigB (15.Lin Y.P. Raman R. Sharma Y. Chang Y.F. J. Biol. Chem. 2008; 283: 25140-25149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). LigBCen2 was shown to bind to both the N-terminal domain (NTD) and the gelatin binding domain (GBD) of Fn. The addition of calcium induces a conformational change in LigBCen2 and enhances binding between LigBCen2 and the NTD of Fn (15.Lin Y.P. Raman R. Sharma Y. Chang Y.F. J. Biol. Chem. 2008; 283: 25140-25149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). microbial surface component recognizing adhesive matrix molecules dynamic light scattering differential scanning calorimetry fibronectin gelatin binding domain isothermal titration calorimetry matrix-assisted laser desorption ionization-time of flight N-terminal domain surface plasmon resonance glutathione S-transferase enzyme-linked immunosorbent assay. microbial surface component recognizing adhesive matrix molecules dynamic light scattering differential scanning calorimetry fibronectin gelatin binding domain isothermal titration calorimetry matrix-assisted laser desorption ionization-time of flight N-terminal domain surface plasmon resonance glutathione S-transferase enzyme-linked immunosorbent assay. The first step in the process of bacterial infection is cellular adhesion, mediated by bacterial adhesins interacting with various components of the extracellular matrix (16.Patti J.M. Allen B.L. McGavin M.J. Höök M. Annu. Rev. Microbiol. 1994; 48: 585-617Crossref PubMed Scopus (932) Google Scholar). Known interaction modes between Fn and bacterial Fn-binding proteins include the β-zipper (17.Schwarz-Linek U. Werner J.M. Pickford A.R. Gurusiddappa S. Kim J.H. Pilka E.S. Briggs J.A. Gough T.S. Höök M. Campbell I.D. Potts J.R. Nature. 2003; 423: 177-181Crossref PubMed Scopus (305) Google Scholar, 18.Bingham R.J. Rudiño-Piñera E. Meenan N.A. Schwarz-Linek U. Turkenburg J.P. Höök M. Garman E.F. Potts J.R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 12254-12258Crossref PubMed Scopus (108) Google Scholar) and the cationic cradle (19.Kingsley R.A. Keestra A.M. de Zoete M.R. Bäumler A.J. Mol. Microbiol. 2004; 52: 345-355Crossref PubMed Scopus (45) Google Scholar). It was recently discovered that the Fn-binding domains in certain Fn-binding proteins are disordered and extended but gain structure upon binding to the NTD of Fn (20.House-Pompeo K. Xu Y. Joh D. Speziale P. Höök M. J. Biol. Chem. 1996; 271: 1379-1384Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 21.Kim J.H. Singvall J. Schwarz-Linek U. Johnson B.J. Potts J.R. Höök M. J. Biol. Chem. 2004; 279: 41706-41714Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 22.Penkett C.J. Redfield C. Jones J.A. Dodd I. Hubbard J. Smith R.A. Smith L.J. Dobson C.M. Biochemistry. 1998; 37: 17054-17067Crossref PubMed Scopus (72) Google Scholar). We have performed a fine-mapping study of the NTD-binding site on LigBCen2 and identified this site as LigBCen2NR, a portion of the non-repeat region (amino acids 1119–1165). The addition of NTD promotes the folding of LigBCen2NR from a disordered and extended structure to a folded structure. This finding is notable, since LigBCen2NR is located in the non-immunoglobulin-like region of LigB, as compared with other Fn-binding proteins, such as Staphylococcus aureus FnbpA and FnbpB (23.Meenan N.A. Visai L. Valtulina V. Schwarz-Linek U. Norris N.C. Gurusidappa S. Hook M. Speziale P. Potts J.R. J. Biol. Chem. 2007; 282: 25813-25902Abstract Full Text Full Text PDF Scopus (80) Google Scholar), Streptococcus dysgalactiae FnBB (17.Schwarz-Linek U. Werner J.M. Pickford A.R. Gurusiddappa S. Kim J.H. Pilka E.S. Briggs J.A. Gough T.S. Höök M. Campbell I.D. Potts J.R. Nature. 2003; 423: 177-181Crossref PubMed Scopus (305) Google Scholar), and Streptococcus pyogenes SfbI and SfbII (24.Schwarz-Linek U. Höök M. Potts J.R. Mol. Microbiol. 2004; 52: 631-641Crossref PubMed Scopus (217) Google Scholar). Thus, the binding mode appears to be similar to the known β-zipper mechanism but unique in sequence-specific interactions. This finding provides the fundamental groundwork for the development of a therapeutic agent to target this interaction in order to prevent or treat Leptospira infection. Rabbit anti-GST antibody and horseradish peroxidase-conjugated goat anti-rabbit antibody were ordered from Molecular Probes, Inc. (Eugene, OR) and Zymed Laboratories Inc. (San Diego, CA), respectively. NTD or GBD of Fn, aldolase, bovine serum albumin, ovalbumin, chymotrypsinogen A, ribonuclease A, aprotinin, insulin chain B, sodium chloride, sodium phosphate monobasic, and sodium phosphate dibasic were purchased from Sigma. The construction, expression, and purification of LigCon (amino acids 1–630) were performed as described previously (12.Palaniappan R.U. Chang Y.F. Jusuf S.S. Artiushin S. Timoney J.F. McDonough S.P. Barr S.C. Divers T.J. Simpson K.W. McDonough P.L. Mohammed H.O. Infect. Immun. 2002; 70: 5924-5930Crossref PubMed Scopus (125) Google Scholar). Constructs for the expression of histidine-tagged or GST-fused LigBCen2 (amino acids 1014–1156) and GST were generated using the vector pQE30 (Qiagen, Alencia, CA) and/or pGEX-4T-2 (GE Healthcare), respectively, as previously described (14.Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar). Constructs for the expression of histidine-tagged or GST-fused LigBCen2R (amino acids 1014–1123) and LigBCen2NR (amino acids 1119–1165) were generated using the vector pQE30 and pGEX-4T-2 (Fig. 1). To perform the PCRs, the following forward and backward primers were utilized (14.Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar): LigBCen2R forward primer 5′-GGATCCACTGCGACTTACAAT-3′ and backward primer 5′-GTCGACCGTGTCCGTTTTGTT-3′; LigBCen2NR forward primer 5′-CGGGATCCAACAAAACGGACACG-3′ and backward primer 5′-CGGTCGACATTGGAACTATTAAT-3′. Primers were engineered to introduce a BamHI site at the 5′-end of each fragment and a stop codon followed by a SalI site at the 3′-end of each fragment. PCR products were sequentially digested with BamHI and SalI and then ligated into pQE30 or pGEX-4T-2 cut with BamHI and SalI, respectively. In this study, we purified the soluble form of the histidine tag or GST fused with LigBCen2, LigBCen2R, or LigBCen2NR from Escherichia coli, as previously described (25.Palaniappan R.U. McDonough S.P. Divers T.J. Chen C.S. Pan M.J. Matsumoto M. Chang Y.F. Infect. Immun. 2006; 74: 1745-1750Crossref PubMed Scopus (108) Google Scholar). Tris buffer (25 mm Tris and 150 mm sodium chloride at pH 7.0) containing 100 μm calcium chloride was used in all experiments, since we have previously shown that calcium enhances the binding of LigBCen2 to NTD (15.Lin Y.P. Raman R. Sharma Y. Chang Y.F. J. Biol. Chem. 2008; 283: 25140-25149Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and that both LigBCen2R and LigBCen2NR bind calcium (data not shown). E. coli harboring the expression plasmid for His-tagged LigBCen2 were cultured in M9 minimal medium. The recombinant LigBCen2 (retaining the His6 tag) was labeled with 15NH4Cl (Cambridge Isotopes, Cambridge, MA), expressed, and purified, as previously described (26.Jayaraman B. Nicholson L.K. Biochemistry. 2007; 46: 12174-12189Crossref PubMed Scopus (16) Google Scholar). The purified 15N-labeled LigBCen2 was dialyzed against Tris buffer with 100 μm calcium chloride at pH 6.0 and concentrated to 0.95 mm (0.3 ml) using the Amicon Ultra centrifugal filter (Millipore, Billerica, MA). For some spectra, 15N-labeled LigBCen2 was also mixed with 1.44 mm (unlabeled) NTD. NMR spectra were recorded at 25 °C on a Varian Inova 600-MHz spectrometer equipped with a triple resonance (hydrogen, carbon, and nitrogen) z axis pulse-field gradient probe. Two-dimensional 15N-1H fast HSQC spectra were recorded with spectral widths of 2.4 kHz in t1 (400 real + imaginary data points) and 10 kHz in t2 (2048 real + imaginary data points) (27.Mori S. Abeygunawardana C. Johnson M.O. van Zijl P.C. J. Magn. Reson. B. 1995; 108: 94-98Crossref PubMed Scopus (569) Google Scholar), with 16 or 32 transients per free induction decay for LigBCen2 in the absence or presence of NTD and a recycle delay of 1.0 s. NMR data were processed and analyzed using the software tools nmrPipe, nmrDraw, and Pipp (28.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11549) Google Scholar, 29.Garrett D. Powers R. Gronenborn A. Clore M. J. Magn. Reson. (1969). 1991; 95: 214-220Crossref Scopus (802) Google Scholar). Data were apodized using a phase-shifted sine bell function and zero-filled prior to Fourier transformation. Association and dissociation rate constants for LigBCen2/NTD binding were measured by SPR analysis performed with a Biacore 2000 instrument (GE Healthcare) at 26.7 °C. 1 μm His-tagged LigBCen2 in Tris buffer containing 100 μm CaCl2 was immobilized on an nitrilotriacetic acid chip (GE Healthcare) conjugated with 500 μm nickel sulfate. Serial concentrations (100, 200, 400, and 800 nm) of NTD were injected into the flow cells at a flow rate of 5 μl/min over the immobilized LigBCen2. All experiments are duplicated. All sensogram data have been corrected by subtracting data from a control cell injected with Tris buffer containing 100 μm CaCl2. Kinetic parameters were obtained by fitting the data to the one-step biomolecular association reaction model (1:1 Langmuir model) with the curve-fitting BIAevaluation software, version 3.0. To determine the binding of GST-LigBCen2, GST-LigBCen2R, GST-LigBCen2NR, or GST-LigCon (negative control) to the NTD of Fn, 1 μm NTD was coated on microtiter plate wells, incubated at 4 °C for 16 h, and blocked with blocking buffer (100 μl/well) containing 3% bovine serum albumin in Tris buffer with 100 μm calcium chloride at room temperature for 2 h. Then serial concentrations (as indicated by Fig. 3A) of GST-LigBCen2, GST-LigBCen2R, GST-LigBCen2NR, or GST-LigCon in 100 μl of Tris buffer with 100 μm calcium chloride were added to the microtiter plate wells for 1 h at 37 °C. To detect the binding of GST-LigBCen2, GST-LigBCen2R, GST-LigBCen2NR, or GST-LigCon, rabbit anti-GST (1:200) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000) were used as primary and secondary antibodies, respectively. After washing the plates three times with TBST (0.05% Tween 20, 100 μm calcium chloride in Tris buffer), 100 μl of TMB (KPL, Gaithersburg, MD) was added to each well and incubated for 5 min. The reaction was stopped by adding 100 μl of 0.5% hydrofluoric acid to each well. Each plate was read at 630 nm using an ELISA plate reader (Bioteck EL-312, Winooski, VT). Each value represents the mean ± S.E. of three trials in triplicate samples. Statistically significant (p < 0.05) differences are indicated by an asterisk. The experiments were carried out with a CSC 5300 microcalorimeter (Calorimetry Science Corp., Lindon, UT) at 25 °C, as described previously (14.Lin Y.P. Chang Y.F. Biochem. Biophys. Res. Commun. 2007; 362: 443-448Crossref PubMed Scopus (67) Google Scholar). In a typical experiment, the cell contained 1 ml of a solution of NTD, and the syringe contained 250 μl of a solution of LigBCen2R or LigBCen2NR. The concentrations of LigBCen2R, LigBCen2NR, and NTD are described in Table 1. Both solutions were in Tris buffer with 100 μm calcium chloride. The titration was performed in 25 injections of 10 μl with a stirring speed of 250 rpm, and the delay time between the injections was 5 min. Data were analyzed using Titration Binding Work version 3.1 software (Calorimetry Science), fitting them to an independent binding model.TABLE 1Thermodynamic parameters for the interaction of NTD and LigBCen2R or LigBCen2NRMacromoleculeLigB residues[LigB][NTD]ΔHTΔSΔGKdnμmμmkcal mol−1kcal mol−1kcal mol−1nmLigBCen2NR1119–11653626113.13 ± 1.5522.25−9.12379 ± 160.94 ± 0.01LigBCen2R1014–11231185.9NFaNF, non-fittable.NFNFNFNFa NF, non-fittable. Open table in a new tab The LigBCen2NR folding prediction was carried out using PONDR (Predictor of Naturally Disordered Regions), a software package containing VL-XT (refers to the merger of three predictors, one trained on variously characterized long disordered regions and two trained on x-ray-characterized terminal disordered regions), XL1-XT (the XL1-XT predictor is a feedforward neural network optimized to predicted regions greater than 39 amino acids), and VL3 (the VL3 predictor is a feedforward neural network that was trained on regions of 152 long regions of disorder that were characterized by various methods), which predict naturally disordered regions (30.Li X. Romero P. Rani M. Dunker A.K. Obradovic Z. Genome Inform. Ser. Workshop Genome Inform. 1999; 10: 30-40PubMed Google Scholar, 31.Romero Obradovic Dunker K. Genome Inform. Ser. Workshop Genome Inform. 1997; 8: 110-124PubMed Google Scholar, 32.Romero P. Obradovic Z. Li X. Garner E.C. Brown C.J. Dunker A.K. Proteins. 2001; 42: 38-48Crossref PubMed Scopus (1365) Google Scholar). PONDR can be used as a Web service for remote and automatic data processing. The analyses were performed using default values. The excess heat capacity Cp(T) of LigBCen2, LigBCen2R, or LigBCen2NR was measured using a DSC Q1000 microcalorimeter (Waters, New Castle, DE). Degassed samples containing 3 μm LigBCen2, LigBCen2R, LigBCen2NR, or Tris buffer with 100 μm calcium chloride were heated at a 10-K/h scan rate. Cp(T) data were recorded, corrected for buffer base line, and normalized to the amount of the samples. The TA Universal Analysis software (Waters) was used for the data analysis and display. All calorimetric experiments in this study were repeated three times to ensure reproducibility. LigBCen2, LigBCen2R, and LigBCen2NR were analyzed for their partition coefficient (Kav) and effective radii (Stokes radii, RS) in Tris buffer with 100 μm calcium chloride using a Superdex 200 HR 10/30 column (GE Healthcare) attached to a fast protein liquid chromatography (GE Healthcare) system. Protein samples were preequilibrated with Tris buffer with 100 μm calcium chloride and eluted with the same buffer at a flow rate of 0.5 ml/min. The column was calibrated using a low molecular weight gel filtration calibration kit (GE Healthcare). The standard globular proteins contained in the kit were ribonuclease A (13,700 Da), chymotrypsinogen (25,000 Da), ovalbumin (43,000 Da), and albumin (67,000 Da). Blue dextran 2000 (2,000,000 Da) (Amersham Biosciences) and aldolase (158,000 Da) were used to indicate the void volume (Vo) and the total volume (Vt), respectively. The elution volume (Ve) of each sample was measured. To define the relationships between the elution volumes of protein samples and their respective molecular weight, the Kav value for each protein was calculated using Equation 1, Kav=Ve−VoVt−Vo(Eq. 1) where Vo and Vt for the column used were 7.96 and 23.56 ml, respectively. Kav values of standard and LigBCen2, LigBCen2R, or LigBCen2NR were plotted against the logarithm of the protein molecular weights to fit Equation 2 as follows (33.Ohno H. Blackwell J. Jamieson A.M. Carrino D.A. Caplan A.I. Biochem. J. 1986; 235: 553-557Crossref PubMed Scopus (23) Google Scholar), logMr=a⋅Kav+b(Eq. 2) where a and b are constants. RS values of the proteins were determined using sample elution volumes and standard curves, as described in the calibration kit (GE Healthcare). One mg/ml LigBCen2, LigBCen2R, or LigBCen2NR was dialyzed against prefiltered (0.22-μm Millipore filters) Tris buffer with 100 μm calcium chloride. The samples were placed in a 1-ml plastic cuvette. The standard globular proteins, including albumin (67,000 Da), ovalbumin (43,000 Da), chymotrypsinogen A (25,000 Da), ribonuclease A (13,700 Da), aprotinin (6,500 Da), or insulin chain B (3,400 Da), were used to generate the calibration curve of globular proteins. The automated measurements were collected with a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Worcestershire, UK), using a 2-min equilibrium delay at each measurement. The data were adjusted using the method of cumulants to obtain the hydrodynamic radii (Rh). The logarithms of the Rh values of standards and LigBCen2, LigBCen2R, or LigBCen2NR were plotted against the logarithm of the protein molecular weights to fit the equation, logMr=a⋅Rh+b(Eq. 3) where a and b are constants. The viscosities of the LigBCen2, LigBCen2R, and LigBCen2NR were measured using a Cannon-Ubbelohde Semi-Micro Dilution Viscometer (catalog number 25 9722-H50; Cannon Instrument Co., State College, PA) with a viscometer constant, 0.002044 mm2/s2, at 25 °C. Before measuring the viscosities, 1 ml of each protein at concentrations of 0.5, 0.75, 1, and 1.5 mg/ml were dialyzed overnight against Tris buffer with 100 μm calcium chloride, with or without 6 m guanidine hydrochloride, and the same buffer was used as a reference solution. The specific viscosity (ηsp) was determined as previously described (34.Tanford C. Kawahara K. Lapanje S. Hooker Jr., T.M. Zarlengo M.H. Salahuddin A. Aune K.C. Takagi T. J. Am. Chem. Soc. 1967; 89: 5023-5029Crossref PubMed Scopus (81) Google Scholar). Specific viscosity/concentration values of untreated or 6 m guanidine hydrochloride-treated LigBCen2, LigBCen2R, or LigBCen2NR were plotted against the concentration of proteins, and the intrinsic viscosity [η] was calculated using the following equation, ηspC=η+kη2C(Eq. 4) where c represents protein concentration and k is a dimensionless constant. The values of [η] expected for a denatured protein shown in Table 3 were obtained from the following equation (34.Tanford C. Kawahara K. Lapanje S. Hooker Jr., T.M. Zarlengo M.H. Salahuddin A. Aune K.C. Takagi T. J. Am. Chem. Soc. 1967; 89: 5023-5029Crossref PubMed Scopus (81) Google Scholar), η=0.716n0.66(Eq. 5) where n represents the number of residues in the protein. CD analysis was performed on an Aviv 215 spectropolarimeter (Lakewood, NJ) under N2 atmosphere. CD spectra were measured at room temperature (25 °C) in a 1-cm path length quartz cell. Spectra of LigBCen2, LigBCen2R, or LigBCen2NR were recorded in Tris buffer with 100 μm calcium chloride at a protein concentration of 10 μm. Three spectra were recorded for each condition from 190 to 250 nm for far UV CD in 1-nm increments. In the thermal denaturation experiment, 10 μm LigBCen2, LigBCen2R, or LigBCen2NR were used, and data were collected at 2 °C/min increments from 20 to 100 °C, recording the ellipticity at 205 nm, with 30-s temperature equilibrations, followed by 30-s data averaging. In order to measure the melting point, th

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