Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Received on December 7, 2000; revised on February 6, 2001; accepted on February 12, 2001.
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Abstract |
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Key words: protein-oligosaccharide complexes/association constants/stoichiometry/Shiga-like toxin/nanoelectrospray ionization
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Introduction |
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The crystal structure of the SLT-1 B5 pentamer complexed with a Gb3 trisaccharide analogue has been solved at 2.8 Å resolution (Ling et al., 1998). Three saccharide binding sites are found in each 7.7 kDa subunit. The subunits associate noncovalently to form a doughnut-shaped B5-pentamer with 15 saccharide binding sites aligned on one face of the toxin. The A subunit is attached to the opposite surface of the B5 pentamer (Stein et al., 1992
; Fraser et al., 1994
), thereby allowing all 15 sites to engage cell surface receptors. Each of the three binding sites interacts with the saccharide moiety of Gb3 in a distinct manner such that sites 1, 2, and 3 bury varying proportions of the trisaccharide epitope (Ling et al., 1998
). The relative importance of the three binding sites observed by crystallography and their contribution toward biological activity is the subject of some speculation. Solution binding studies by titration microcalorimetry (St. Hilaire et al., 1994
) or by nuclear magnetic resonance (NMR) studies (Shimizu et al., 1998
) suggest that site 2 provides the crucial recognition, with minor contributions from sites 1 and 3. Based on computer modeling studies and receptor analogue binding data, site 1 was concluded to be the major Gb3 binding site (Nyholm et al., 1996
). The involvement of multiple sites was invoked on the basis of mutagenesis and crystal structure data (Ling et al., 1998
; Bast et al., 1999
).
We recently reported the design, synthesis, and crystal structure of a multivalent subnanomolar inhibitor complexed with SLT-1 B5 pentamer (Kitov et al., 2000). The inhibitor design based on the published crystal structure placed two trisaccharide receptors at the tips of each of five spacer arms to permit the simultaneous engagement of sites 1 and 2 in all five B subunits. Although the inhibitor successfully embraced the toxin surface and placed the Pk trisaccharides in each B subunit, the bivalent ligand at the end of each arm occupied only site 2 of a single B subunit and does not bridge sites 1 and 2 as planned. Instead the remaining five copies of the Pk trisaccharide complexed a second molecule of toxin again via saccharide receptor binding to site 2.
During studies of oligosaccharide binding to single chain antibody using nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), we realized that this technique could provide unique data on the occupancy of the different binding sites of SLT-1. We demonstrate here the application nanoES/FT-ICR MS to delineate aspects of the self-association of the multimeric protein, SLT, as well as the stoichiometry and affinity of its specific association with various analogs of its natural receptor ligand, globotriaose [-D-Galp (1
4)ß-D-Galp (1
4)ß-D-Glcp], also known as Pk trisaccharide.
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Results |
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Discussion |
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The difficulty in applying ES-MS to such weakly interacting complexes is the high analyte concentration required to produce a significant amount of complex in solution. High concentrations of analyte in the ES solution often leads to observation of nonspecific complexes, resulting from the formation of random intermolecular interactions as the ES droplet shrinks due to evaporation of solvent. The presence of such complexes in the mass spectrum obscures the true solution composition. In addition to the possibility of the formation of nonspecific complexes, decomposition of proteinoligosaccharide complexes by collisional heating, which occurs during sampling into the mass spectrometer, may also influence the spectrum. The effects of in-source dissociation will be most significant for complexes that are weakly bound in the gas phase and prone to facile decomposition. The fact that quantitative agreement was observed between the solution composition and the relative ion abundance in the mass spectra suggests that neither nonspecific binding nor decomposition of the complexes in the gas phase was significant. The presence of nonspecific binding is normally identified by the disappearance of complexes on reduction of the analyte concentration. In the present case, the dissociation constant determined from the mass spectrometric data was found to be independent of the concentration of 1, which was varied from 45 to 310 µM. Therefore, if nonspecific complexes are produced by the nanoES process, they do not survive long enough to be detected.
We have also investigated the thermal decomposition kinetics for B5·(Pk)n complexes using the blackbody infrared radiative dissociation (BIRD) technique (Price et al., 1996). This study (Felitsyn et al., unpublished data) revealed that the complexes are quite robust in the gas phase, presumably due to the retention of the proteinsugar hydrogen bonds originally present in solution and, perhaps, the formation of additional interactions. For example, at relatively high temperatures (
170°C) loss of a subunit is more facile than loss of Pk from protonated B5·(Pk)n ions. Therefore, Pk trisaccharides that are bound specifically are unlikely to be lost during introduction into the mass spectrometer. However, B5·Pk complexes that originate from nonspecific binding, via the formation of random interactions during the desolvation process, may be much more labile in the gas phase and easily decomposed during sampling. If this is the case, these weakly bound complexes may be filtered out in the source, leaving only the more stable, specific complexes. In an effort to address this question, we are extending our BIRD studies to evaluate the stability of other proteinoligosaccharide complexes.
The mass spectrometric data obtained for SLT-1(B5) and 1 allowed us to establish that the binding sites of each subunit operate in a noncooperative manner and that preferential occupation of all five copies of the toxin site 2 occurs prior to occupancy of site 1. The relative importance of the three different binding sites has been a matter of debate in the literature. Our results, which indicate the presence of only one major binding site per subunit, are in agreement with the solution NMR studies (Shimizu et al., 1998). Furthermore, based on the relative abundance of bound and unbound B5, we were able to determine the binding constant for attachment of the first five Pks at site 2. The mass spectrometric data also suggest that the Pk affinity for site 2 is 10-fold higher than for site 1.
NanoES/FT-ICR MS is an exquisite method to study the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as SLTs. It permits the facile identification of the occupancy of binding sites, information that is not readily available by other techniques. Its high-resolution capability is ideally suited to the observation of interactions between a large protein receptor and a relatively small oligosaccharide ligand. The sensitive and rapid estimation of association constants for proteinligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.
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Materials and methods |
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The trimethylsilylethyl glycoside of Pk trisaccharide 1 was synthesized as described by Kihlberg et al. (1989) and purified by reverse phase high-performance liquid chromatography. Stock solution with 2 mg/ml concentration in deionized water was used. Synthesis of divalent tethered compound 2 and the decavalent STARFISH molecule 3 will be described elsewhere (Kitov and Bundle, submitted).
Mass spectrometry
Mass spectra were obtained using a Bruker 47e ApexII FT-ICR mass spectrometer equipped with electrospray source (Analytica, Branford). To facilitate the observation of weakly bound noncovalent complexes, the glass sampling capillary in the source was replaced with a stainless steel desolvation capillary (0.43 mm i.d.) which was operated at 150°C. NanoES was performed using aluminosilicate capillaries (0.68 mm i.d.), pulled to approximately 20 µm o.d. and 110 µm i.d. at one end using a micropipette puller (Sutter Instrument Co.). The nanospray tips were positioned approximately 1 mm from the sampling capillary using a microelectrode holder (Warner Instrument Inc.). The electric field required to spray the solution was established by applying a voltage of 8001000 V to a platinum wire inserted inside glass tip. The solution flow rate ranged from 5 to 60 nl/min depending on diameter of the nanoelectrospray tip, electrospray voltage, and composition of the solution.
The droplets formed at atmospheric pressure in the interface between the nanoES tip and the metal capillary were introduced to the vacuum system of mass spectrometer. Solvent evaporation from the droplets or microsolvated complexes was assisted by thermal heating provided by sampling capillary. The ion/gas jet sampled by the capillary (52 V) was transmitted through a skimmer (4 V) and stored, electrostatically, in the hexapole. Ions were accumulated in hexapole for 215 s, depending on the ion intensities, then ejected and accelerated by using high potential (2700 V) through the fringing field of the 4.7 Tesla magnet, decelerated, and introduced into the ion cell and detected.
The ion intensities observed for the toxinoligosaccharide complexes were found to be very sensitive to electrospray and source conditions (nanospray tip dimensions, solution flow rate, temperature of metal capillary, applied voltages, and storage time ions in hexapole). The strongest ion signals for complexes were observed at a relatively low electrospray voltage (800 V) and a low solution flow rate (
510 nl/min). Increasing the solution flow rate required longer storage times in hexapole (up to 15 s) to generate comparable ion abundances.
Data acquisition was controlled by SGI R5000 computer running the Bruker Daltonics XMASS software version 5.0. Mass spectra were obtained using standard experimental sequences with chirp broadband excitation. The time-domain signal, consisting of the sum of 10500 transients containing 128 K data points per transient, was subjected to one zero-fill prior to Fourier transformation. The number of transients acquired varied in different experiments depending on signal-to-noise ratio.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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