1 Pharmacogenetic Section Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
2 Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
3 Department of Bacteriology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
Correspondence
Lars C. Pedersen
pederse2{at}niehs.nih.gov
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ABSTRACT |
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These authors made equal contributions to this work.
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INTRODUCTION |
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To better understand how type A HA1 interacts with sugars and thus may be involved in internalization of the neurotoxin, the techniques of isothermal titration calorimetry (ITC) and protein crystallography have been employed. Although we were unable to obtain diffraction quality crystals of type A HA1, we were able to crystallize and determine the X-ray structure of type C HA1. Type C HA1 is 37 % identical in sequence to type A HA1 and thus should provide a good structural model for type A HA1. Type C HA1 consists of two -trefoil domains and bears structural similarities to the crystal structure of the ricin B-chain with lactose bound. ITC analysis was successfully applied to characterize carbohydrate-binding specificity of type A HA1, suggesting a single binding site with preference for galactose (Gal) and oligosaccharides containing Gal at the non-reducing end. Based on the sequence alignment of type A HA1 to type C HA1 and the structural comparison of type C HA1 to the ricin B-chain lactose-binding sites, site-directed mutagenesis was performed on type A HA1 to determine the carbohydrate-binding site. ITC analysis of these mutants revealed that the D263A and N285A mutants completely abrogated carbohydrate binding, implicating these residues in carbohydrate binding.
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METHODS |
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The HA1GST fusion protein was expressed in Escherichia coli BL21(DE3) cells (Invitrogen). The transformed bacteria were grown in LuriaBertani (LB) medium containing 100 µg ampicillin ml-1 at 37 °C. Growth was monitored by optical density measurements at 600 nm; at an OD600 value of 0·8, expression was induced with 0·1 mM IPTG. The cells were grown overnight at room temperature in 18 l of LB medium and harvested by centrifugation (5000 g, 20 min). Cells were resuspended in PBS, sonicated and spun down at 38 000 r.p.m. for 30 min at 4 °C. The supernatant was collected and incubated with Glutathione Sepharose 4B (Amersham Pharmacia Biotech) resin (1 ml resin per 1 l induced cell culture), equilibrated with PBS and shaken gently for 30 min at 4 °C. The resin was washed five times in batch with a volume of PBS 25 times that of the resin for each wash. The fusion protein was cleaved on the resin with 50 units of thrombin (Sigma) overnight at room temperature. The supernatant/Sepharose mixture was poured into a column with a fritted filter and the resin was washed with PBS until the OD280 value became less than 0·1. The eluted solution was applied to a Benzamidine Sepharose 6B (Amersham Pharmacia Biotech) column to remove thrombin. The resulting solution was collected and then applied onto a Ni-NTA agarose (Qiagen) column pre-equilibrated with 20 mM Tris/HCl (pH 7·5) containing 0·1 M NaCl. After washing the column with equilibration buffer, bound protein was eluted using a 0500 mM imidazole gradient. Fractions that contained pure HA1 were pooled, dialysed against 20 mM MES (pH 5·5), concentrated to 25 mg ml-1 and stored at -80 °C.
Expression and purification of type A HA1.
The expression vector pGEX-5X-3 type A HA1 constructed previously (Fujinaga et al., 2000) was used as a template. Primers 5'-CCGCGTGGATCCATGGAACACTATTCAGTAATC-3' and 5'-CGGCCGCTCGAGTTATGGGTTACGAATATTCCA-3' were designed for amplification with PCR. The product was restricted with BamHI and XhoI and, after purification by agarose gel electrophoresis, was inserted into the expression vector pGEX-4T-3 and transformed into E. coli BL21(DE3) cells. Using a similar protocol as for type C HA1, the protein was expressed and then purified on Glutathione Sepharose 4B resin. The protein was eluted from the resin by overnight thrombin cleavage (6 units per millilitre of resin). The eluted protein was then dialysed overnight against 10 mM phosphate buffer, pH 5·5. All type A HA1 mutants were purified using the same protocol as the wild-type protein.
Site-directed mutagenesis of type A HA1.
Site-directed mutagenesis was performed using the QuickChange Site-Directed Mutagenesis kit (Stratagene) following the protocols described in the accompanying instruction manual. The primers used were as follows: for D171A mutation, 5'-GTCGTACAACAAGTGGCTGTGACAAATCTAAAT-3' and 5'-ATTTAGATTTGTCACAGCCACTTGTTGTACGAC-3'; for N187A mutation, 5'-TGGGACTATGGTCGCGCTCAAAAATGGACAATT-3' and 5'-AATTGTCCATTTTTGAGCGCGACCATAGTCCCA-3'; for D263A mutation, 5'-ACAACTAAAGCTCTAGCTTTATATGGCGGCCAA-3' and 5'-TTGGCCGCCATATAAAGCTAGAGCTTTAGTTGT-3'; for N285A mutation, 5'-TATCATGGAGATGATGCTCAGAAATGGAATATT-3' and 5'-AATATTCCATTTCTGAGCATCATCTCCATGATA-3'. Sequences were confirmed, following mutagenesis, with the Big Dye Terminator Cycle Sequencing Reaction Kit (Applied Biosystems) in accordance with the protocol found in the accompanying instruction manual.
ITC.
ITC measurements were carried out using a VP-ITC MicroCalorimeter (Micro Cal) at 30 °C. Solutions containing carbohydrate concentrations of 60 mM were injected into a reaction cell containing about 200 µM protein. Thirty injections of 6 µl at 180 s intervals were performed. Data acquisition and analyses were performed using the MICROCAL ORIGIN software package.
Crystallization of type C HA1.
Crystals of type C HA1 were obtained using the vapour-diffusion hanging-drop method. Purified protein was mixed with equal volumes of a reservoir solution consisting of 0·1 M MES (pH 5·5), 100 mM MgCl2 and 10 % PEG-8000 and placed above the reservoir. For data collection, harvested crystals were transferred to a cryoprotectant consisting of 0·1 M MES (pH 5·5), 100 mM MgCl2, 12 % PEG-8000 and 15 % ethylene glycol and then flash-frozen at the detector in a stream of nitrogen cooled to -170 °C. Crystals were transferred to the cryosolution in four steps of increasing ethylene glycol and PEG concentration. For the HgCl2 derivatized crystal, the crystal was soaked in the reservoir solution containing saturated HgCl2 (<1 mM) overnight then back-soaked into the same cryoprotectant used for native crystals.
The higher resolution native dataset (native 2) was collected from a crystal soaked in the 11 mM tetra-saccharide lacto-N-neo-tetraose; however, no electron density was detected for the tetra-saccharide. All datasets were collected with an RAXIS IV area detector system with Yale/MSC mirrors using a RU3H Rigaku generator. Data were indexed and integrated with DENZO and scaled using SCALEPACK (Otwinowski & Minor, 1997) (Table 1
). Phases were obtained using the single isomorphous replacement with anomalous scattering (SIRAS) technique. SHELX was used to determine the position of the heavy metal sites (Sheldrick et al., 1993
), MLPHARE was used to calculate phases, and DM was used to improve the quality of the electron density map (Bailey, 1994
). Automated model building using WARP produced half of the model (Perrakis et al., 1999
). Two molecules were found in the asymmetric unit. Both molecules contain electron density for residues 4286 out of the 286 expected and both have good geometry as determined by PROCHECK (Laskowski et al., 1993
). All programs, with the exception of DENZO and SCALEPACK, were from the CCP4 interactive package (Bailey, 1994
).
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RESULTS AND DISCUSSION |
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The -trefoil domain is a structural fold found in other proteins including lectins such as the ricin B-chain (Rutenber et al., 1987
) and Amaranthus caudatus agglutinin (ACA) (Transue et al., 1997
), cytokines such as fibroblast growth factor (FGF) (Faham et al., 1996
; Ornitz et al., 1995
; Zhang et al., 1991
) and interleukin-1 (IL-1) (Finzel et al., 1989
), Kunitz trypsin inhibitors such as that from soybean (Sweet et al., 1974
), the actin cross-linking protein hisactophilin (Habazettl et al., 1992
), the xylan-binding domain of xylanase 10A (XBD-10A) (Notenboom et al., 2002
), the cysteine-rich domain of the mannose receptor (Liu et al., 2000
), and even in the BoNT structures (Lacy et al., 1998
; Swaminathan & Eswaramoorthy, 2000
). In many instances, the
-trefoil domain acts as an oligosaccharide-binding unit supporting the suggestion that the
-trefoil fold itself is a result of a gene triplication of an ancient carbohydrate-binding peptide (the four-strand trefoil motif) of approximately 40 residues (Rutenber et al., 1987
).
Position, orientation and interactions between -trefoil domains and carbohydrates vary among different proteins. In the case of ligand binding to the BoNT/B protein, the majority of the interactions between the protein and the sialylactose ligand are with the sialic acid substituent (Swaminathan & Eswaramoorthy, 2000
). Binding of a substituent also dictates carbohydrate binding to the cysteine-rich domain of the mannose receptor where the majority of the interactions between ligand and protein are backbone amides forming hydrogen bonds with a sulfate moiety (Liu et al., 2000
). In addition, sulfate moieties are responsible for heparan sulfate binding to the FGF1
-trefoil protein. In the crystal structure of the FGF1FGFR2heparan sulfate complex, the heparan sulfate molecule binds two FGF1s to create the FGF1 dimer required for binding to the FGFR2 (Pellegrini et al., 2000
). The non-toxic plant lectin ACA, like HA1, contains two
-trefoils per subunit. This protein is a dimer in solution even when carbohydrate is not bound. ACA contains two sugar-binding sites specific for the T-disaccharide [
-D-gal-(1
3)-D-GalNAc] and related by dimer symmetry. The 1
- and 1
-repeats from one molecule and the 2
-repeat from the dimer-related molecule make contacts with the flat sides of the sugars to make up one binding site (Transue et al., 1997
).
Lactose binding to -trefoil domains has been observed in the ricin B-chain and the XBD-10A structures (Notenboom et al., 2002
; Rutenber & Robertus, 1991
). Like HA1 and ACA, the ricin B-chain comprises two
-trefoil domains. In this structure, each of the
-trefoil domains (1
and 2
sites) binds one lactose molecule (Rutenber & Robertus, 1991
). In both cases the Gal moiety forms hydrogen bonds with an Asp from
-strand 2 of the repeat, an Asn from just before
-strand 4 of the repeat, and forms van der Waals contacts with an aromatic residue from
-strand 3 of the repeat. Similar binding is also seen with lactose binding to XBD-10A (Notenboom et al., 2002
). The XBD-10A structure is a single
-trefoil that binds lactose by the Gal sugar at the
and
positions in a manner similar to lactose binding to ricin B-chain. Interestingly, the reducing glucose sugar protruding from the
-repeat is seen binding to the
-repeat of a crystal symmetry-related XBD-10A molecule. Thus, lactose is found binding at all three sites in the crystal structure.
ITC analysis of carbohydrate binding to type A HA1
Previous work demonstrated that lactose inhibited haemagglutinin activity of type A HA1(Inoue et al., 2001), therefore ITC was utilized to investigate lactose binding to type A HA1. The heat released was measured during successive injections of lactose solution, and the integrated heats of binding were plotted against the molar ratio of lactose and type A HA1 to generate a binding isotherm. From this, the stoichiometry, affinity (dissociation constant Kd), entropy (
S) and enthalpy (
H) of the binding were determined. Heat was released as an indication of the binding of
-lactose to the type A HA1 protein (Fig. 2
a). However, no heat was released either when lactose solution was successively injected into the buffer solution without type A HA1 (Fig. 2b
) or when BSA was used instead of type A HA1 (data not shown). The stoichiometry of the heat release suggests that type A HA1 has a single binding site per molecule.
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Received 20 June 2003;
revised 29 August 2003;
accepted 2 September 2003.
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