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Structure of trypanosome coat protein VSGsur and function in suramin resistance

Nature Microbiology volume 6, pages 392–400 (2021)Cite this article

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Abstract

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

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Fig. 1: Overall structure of VSGsur.
Fig. 2: Suramin binds only VSGsur and is more toxic to all other VSGs examined.
Fig. 3: Suramin binds in the cavity between the VSGsur monomers.
Fig. 4: VSGsur and the loss-of-contact mutants have indistinguishable endocytic kinetics.
Fig. 5: Model for possible mechanism of VSGsur-mediated resistance to suramin.

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Data availability

Coordinates and structural factors have been uploaded to the RCSB PDB (www.rcsb.org): VSGsur I3C (PDB ID 6Z79), VSGsur WT native (PDB ID 6Z7A), VSGsur + suramin (PDB ID 6Z7B), VSGsur H122A (PDB ID 6Z7C), VSGsur H122A 0.77 mM suramin (PDB ID 6Z7D), VSGsur H122A 7.7 mM (PDB ID 6Z7E), VSG13 NaBr (PDB ID 6Z8G6Z7G) and VSG13 native (PDB ID 6Z8H). Other data supporting the findings of this study are available from the authors upon request. Source data are provided with this paper.

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Acknowledgements

We acknowledge time at the European Synchrotron Radiation Facility (ESRF, beamline ID29, proposal MX1975, G. Santoni and colleagues), the Diamond Light Source (DLS, beamline i03, proposal number mx18989-1, N. Paterson and colleagues), the Helmholtz-Zentrum Berlin (BESSY, beamlines 14.1 and 14.2, proposal numbers MX-191-00036 and MX-192-00114, staff member M. Weiss and colleagues, especially C. Feiler for help with processing and solving the I3C SAD dataset) and the Paul Scherrer Institut, Villingen, Switzerland (SLS, beamline X06DA, proposal numbers 20182345, 20191097 and 20191895, V. Olieric and colleagues). We thank L. Figueiredo for supplying us with the VSG13-expressing T. brucei strain, and M. Cal, R. Rocchetti, S. Keller and M. Kaiser for help with drug sensitivity testing. N.W., S.H. and P.M. were supported by the Swiss National Science Foundation (grant no. 310030_185163). The work was supported by funds and resources from the German Cancer Research Center (DKFZ).

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Author notes

  1. These authors contributed equally: Johan Zeelen, Monique van Straaten.

Authors and Affiliations

  1. Division of Structural Biology of Infection and Immunity, German Cancer Research Center, Heidelberg, Germany

    Johan Zeelen, Monique van Straaten, Joseph Verdi, Alexander Hempelmann & C. Erec Stebbins

  2. Division of Immune Diversity, German Cancer Research Center, Heidelberg, Germany

    Joseph Verdi, Hamidreza Hashemi & F. Nina Papavasiliou

  3. Protein Expression and Purification Core Facility, EMBL Heidelberg, Heidelberg, Germany

    Kathryn Perez

  4. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Philip D. Jeffrey

  5. Swiss Tropical and Public Health Institute, Basel, Switzerland

    Silvan Hälg, Natalie Wiedemar & Pascal Mäser

  6. University of Basel, Basel, Switzerland

    Silvan Hälg, Natalie Wiedemar & Pascal Mäser

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Contributions

H.H. cloned VSGsur and M.v.S. produced VSGsur mutants. S.H., N.W. and P.M. produced high-resistance VSGsur mutants and associated assays. M.v.S. and J.Z. purified all VSGs. M.v.S. crystallized VSG13. M.v.S. and J.Z. crystallized wild-type and mutant VSGsur proteins. C.E.S. phased and produced the first model of VSG13 and A.H. built and refined the structures of VSG13 native and the sodium bromide soak. J.Z. solved all crystal structures of VSGsur. P.D.J. worked with J.Z. on the refinement of VSGsur bound to suramin. M.v.S. performed the suramin growth and toxicity assays. K.P. conducted ITC experiments. J.V. conducted endocytosis assays.

Corresponding author

Correspondence to C. Erec Stebbins.

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The authors declare no competing interests.

Additional information

Peer review information Nature Microbiology thanks Raghuvir Arni, Markus Engstler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Comparison of the VSG NTDs.

a, A single monomer of the VSGsur NTD is shown as a ribbon diagram. The β-sheet subdomain that forms the top lobe is colored in orange and the rest of the structure in light blue. The N-linked glycan is shown as red space-filling spheres, the cysteine disulfide atoms as space-filling cyan spheres. b, Structural alignment of monomers of VSGsur (orange) and VSG13 (grey) with corresponding glycans shown in space filling representation and colored the same as the protein to which they are linked, while disulfides are shown in cyan and green for VSGsur and VSG13, respectively. The alignment produces an overall root-mean-square deviation of 1Å for the conserved portions of the structure (calculated over 220 Cα positions that primarily encompass the three-helix bundle and elements of the bottom lobe). c, Comparison of the structures of VSGsur, VSG13, and VSG2. The N-linked glycans are displayed as red space-filling atoms and the disulfide bonds are shown in cyan. Approximate dimensions of the molecules are noted, as well as the directions toward the external environment and toward the C-terminal domain (CTD) and plasma membrane of the trypanosome.

Extended Data Fig. 2 Growth curves of VSGsur and VSG mutants.

Strains expressing VSGsur and VSGsur mutants were grown with and without suramin (incubation of 0.7µM suramin for 24 h, Methods). The cell densities were determined by cell counting using a Neubauer hemocytometer. This procedure was repeated for 3-4 days in a row. Analysis was performed with GraphPad Prism, using a nonlinear regression model for curve fitting (Exponential growth with log). For statistical comparisons, we tested whether the slopes and intercepts of the best fit curves were significantly different. For VSGsur and VSGsur + 0.7µM suramin, the differences between the slopes is not significant (P value = 0.7720). For H122A and N130A (without suramin) compared to VSGsur the differences are significant (P<.0113 and P<.0001, respectively). Two independent experiments with different cultures were made for each data point.

Extended Data Fig. 3 Sample ITC results.

ITC data for suramin binding to each VSG protein. The upper panels contain the baseline corrected raw data, and the lower panel contains the peak-integrated, concentration normalized data for the heat of reaction vs. molar ratio of suramin per VSG protein. a, VSGSur was measured 3 times, independently: 300µM suramin was titrated into 46µM VSGsur, the curve fitted with a single binding site model to calculate a Kd of 234+/- 28 nM and N of 0.49 +/- 0.03 b, VSGSur H122A was measured 3 times, independently: 450µM suramin was titrated into 51.1µM VSGSur H122A. A Kd could not be fit to the data, although it is clear that the mutation negatively affected the binding affinity. c, VSGSur N130A was measured 2 times, independently: 300µM suramin was titrated into 40µM VSGSur N130A. No binding was detected. d, VSG2 was measured 2 times, independently: 200µM Suramin was titrated into 20µM VSG2 protein. No binding was detected. e, VSG3 was measured 2 times, independently: 300µM suramin was titrated into 53.1µM VSG3. No binding was detected. f, VSG13 was measured 2 times, independently: 200µM Suramin was titrated into 20µM VSG132 protein. No binding was detected.

Extended Data Fig. 4 Two-fold axis of dimer symmetry for VSGsur-suramin complex.

a, The top drawing illustrates the chemical structure of suramin with several of its functional groups denoted, whereas the bottom renders the drug as found in the protein structure with a transparent molecular surface shown. Oxygen atoms are shown in red, nitrogen in blue, and carbon gray. b, Ribbon diagram of VSGsur (one monomer blue and the other gold) looking down the two-fold axis of symmrtry for the dimer. Suramin is shown as a ball-and-stick chemical representation as in (a). In the center of the rotational axis for the dimer, one of the suramin benzene rings is visible.

Extended Data Fig. 5 H122 “open” and “closed” conformations.

a, Two H122 conformations in the native crystals structure of VSGsur with corresponding electron density (b) Closed conformation electron density of H122 (c) Closed conformation surface of VSGsur (d) Position of H122 and the I3C group used in phasing the crystal structure with corresponding electron density.

Extended Data Fig. 6 Creation of VSGsur and VSGsur mutant expressing strains.

The same construct was used multiple times to generate different VSG-expressing cell lines. The top schematic shows the endogenous sub-telomeric expression site in 2T1 cells, which express VSG2 (pink) with its endogenous UTRs (dark green). The vector used to integrate new VSGs was adapted from Pinger et al.2, wherein the homology directed integration of a novel VSG ORF (black) flanked by the UTRs of VSG2 is mediated by 3’ telomere seeds (Tel-S) and 5’ homology to the upstream co-transposed region (CTR – teal). Transfected cells are identified by screening for the integration of a hygromycin selection cassette (red) that is expressed via read-through transcription driven by the subtelomeric promoter.

Extended Data Fig. 7 Panels of WT and H122A with suramin soaks.

Molecular surface of one monomer in the VSGsur dimer shown colored by electrostatic potential (white is neutral, blue is positive/basic, and red is negative/acidic in charge). Electron density in the suramin binding site is shown in green.

Extended Data Fig. 8 Resistance enhancing mutations mapped to VSGsur.

Ribbon diagrams of VSGsur/suramin co-crystal structure in two orientations. Mutations discovered in “supersur” VSGsur mutants with heightened resistance to suramin are shown as green space filling atoms. Suramin and the N-linked glycan of VSGsur are depicted as space filling atoms in purple and crimson respectively.

Extended Data Fig. 9 Replacement of VSGsur with "supermutant" VSGsur at the active expression site of T. b. rhodesiense STIB900_sur1.

a, Construct for gene replacement; mutations in the VSGsur coding sequence (dark red) are shown in light red, arrows indicate primer binding sites. BSD, blasticidin resistance gene; αβ tub, αβ tubulin splice site. (b) 50% inhibitory concentrations of the transfected clones and the parent (sur1) as measured with Alamar Blue assays. The clones sur1_tr1 and sur1_tr2 still expressed VSGsur, while the clones sur1_tr3 and sur1_tr4 expressed the mutant version. The scatter plots represent independent drug assays, each carried out in duplicate. Error bars represent mean ± standard deviation. Small letters indicate significance groups as determined by one-way ANOVA (Df = 4, F-value = 31.02, p-value = 3.6×10−9), followed by Tukey’s multiple pairwise comparisons test (p-values <0.001) as calculated with R 3.6.2. n = 6 for STIB900_sur1, sur1_tr1, sur1_tr2, and sur1_tr4; n = 5 for sur1_tr3.

Source data

Extended Data Fig. 10 Endocytosis rates by different VSGs relative to VSG2.

Alexa 488-dextran (a), bodipy-LDL (b), and Alexa 488-transferrin (c) endocytosis by T. b. brucei 2T1 cells expressing a variety of different VSG genes. Due to technical limitations, each cell line could not be analyzed simultaneously within each experiment for each ligand. Therefore, VSG2 was used as a control in all experiments, allowing the calculation of each cell line’s relative uptake rate of each ligand as compared to the uptake rate of each ligand observed by VSG2 expressing cells within each separate experiment. Each of these graphs therefore represent the combined experimental results from 2 separate experiments. Cell lines with a relative fluorescence intensity below 1 at a given time point have less efficient endocytic rates compared to VSG2 expressing cells, and vice versa. All graphs share the same Y axis.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Discussion, Methods and Tables 1–4.

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Source data

Source Data Fig. 2

The first table gives the cell densities of Trypanosoma strains expressing different VSGs, measured 24 h after addition of various concentrations of suramin. The starting density was 100 (×1,000) cells per ml. (Cells were grown in 24-well tissue culture plates. Cell numbers were determined with a Neubauer haemocytometer.) In the second table, the values are normalized to the cell growth without suramin. (No suramin is 100%.)

Source Data Extended Data Fig. 9

The Excel file contains the data for Extended Data Fig. 9. It is organized in five worksheets, one for each T. b. rhodesiense line: the parental STIB900_sur1 and the four transfected descendants thereof (sur1_tr1 to sur1_tr4). Each line was tested against the drugs melarsoprol (rows 2 to 17) and suramin (rows 19 to 34). Drug concentration in nanomolar is in column A; percentage of growth as compared with untreated controls, tested in six replicates, is in columns B to G. The resulting dose–response curves are shown on the right side. The calculated pharmacodynamic parameters are in rows 14 to 17 for melarsoprol and in rows 31 to 34 for suramin. The IC50 values as plotted in Supplementary Fig. 11 are labelled in green (row 32). Note that melarsoprol was used as an internal reference; the data are not included in Supplementary Fig. 11.

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Zeelen, J., van Straaten, M., Verdi, J. et al. Structure of trypanosome coat protein VSGsur and function in suramin resistance. Nat Microbiol 6, 392–400 (2021). https://doi.org/10.1038/s41564-020-00844-1

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