Figure 4. Simultaneous binding of two receptors to each HpHb dimer leads to more efficient uptake into trypanosomes.

(A) A model for a complex of one HpHb dimer bound to two receptors, generated by docking the structure of the TbHpHbR:HpSPHb complex onto that of porcine HpHb (Andersen et al., 2012). The receptors are organized such that two receptors, both associated with the membrane through attachment at their C-termini, can simultaneously bind to one HpHb dimer. (B) An ab initio molecular envelope derived from small angle x-ray scattering analysis of the TbHpHbR:HpHb complex supports the formation of a complex containing one HpHb dimer bound to two receptors. (C) Uptake of fluorescently labelled dimeric HpHb into live cells was monitored via flow cytometry across a range of 1–62.5 nM. Uptake saturated by 4 nM in wild-type cells whereas no uptake was observed in the HpHbR null cell line. No fluid phase uptake of labelled BSA was observed at these concentrations. (D) Uptake of fluorescently labelled monomeric HpSPHb was not readily detected until 62.5 nM, at which point uptake had not saturated. HpSPHb uptake at 62.5 nM was lost in the HpHbR null cell line. Each uptake assay was carried out in triplicate. Error bars represent standard error of the mean, n = 3.

DOI: http://dx.doi.org/10.7554/eLife.05553.016

Figure 4—figure supplement 1. Small angle x-ray scattering of HpHb, alone and in complex with TbHpHbR.

(A) An ab initio molecular envelopes calculated from scattering data from the HpHb complex. (B) The theoretical scattering calculated from ab initio reconstructions for HpHb superimposed into experimental scattering data. Guinier plots are shown as an insert. (C) Distance distribution functions of HpHb derived from small angle x-ray scattering. (D) An ab initio molecular envelopes calculated from scattering data from the TbHpHbR:HpHb complex. (E) The theoretical scattering calculated from ab initio reconstructions for TbHpHbR:HpHb superimposed into experimental scattering data. Guinier plots are shown as an insert. (F) Distance distribution functions of TbHpHbR:HpHb derived from small angle x-ray scattering.

DOI: http://dx.doi.org/10.7554/eLife.05553.017

Figure 4—figure supplement 2. SEC MALLS data to assess the stoichiometry of the TbHpHbR:HpHb complex.

Multi-angle light scattering (MALLS) measurements of TbHpHbR (red), HpHb (blue) and the TbHpHbR:HpHb complex (green). The molecular weights determined from scattering data (∼30 kDa for TbHpHbR, ∼150 kDa for HpHb and ∼210 kDa for the TbHpHbR:HpHb complex) show the formation of a complex containing two receptors bound to a single HpHb.

DOI: http://dx.doi.org/10.7554/eLife.05553.018

Figure 4—figure supplement 3. Establishment and characterization of an HpHb^−/− cell line of T. brucei.

TbHpHbR null cell lines were generated in T. b. brucei Lister 427 bloodstream form (BSF) cells. (A) The TbHbHbR gene was knocked out in Lister 427 BSF cells by replacement of one allele with a blasticidin resistance gene and the other allele with a neomycin resistance gene in four independent clones, as confirmed by Southern blot (P = Parental cell line, 1–4 = TbHpHbR null clones 1–4). The schematic depicts the original (top) and replacement (middle and lower) TbHpHbR loci and the positions of Southern blot probes and restriction enzyme sites used. Expected fragment sizes are annotated. (B) Growth of the TbHpHbR null clones was monitored in vitro over 192 hr. Parental L427 cells grew with a mean doubling time of 8.4 hr whereas the TbHpHbR null clones had an increased mean doubling time of 11.5–12.0 hr. Error bars represent standard deviation, n = 8.

DOI: http://dx.doi.org/10.7554/eLife.05553.019