Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor
- University of Oxford, United Kingdom
- University of Cambridge, United Kingdom
Figures
Figure 1 with 2 supplements
The structure of the T. brucei haptoglobin-haemoglobin receptor.
(A) The structure of the T. brucei haptoglobin-haemoglobin receptor, with helix I (red), helix II (orange) and helix V (blue). These three helices form an elongated bundle with a ∼50° kink towards …
Figure 1—figure supplement 1
Surface plasmon resonance analysis of the binding of HpSPHb to TbHpHbR.
Surface plasmon resonance signals for twofold dilutions of HpSPHb complex from a maximum concentration of 16 μM, binding to a surface coated with the truncated version of T. brucei HpHbR.
Figure 1—figure supplement 2
Alignment of the TbHpHbR and TcHpHbR structures.
Structural alignment of T. brucei HpHbR (blue) with T. congolense HpHbR (red). The membrane distal (upper) halves of the receptors align with a root mean square deviation of 1.1 Å while the membrane …
Figure 2 with 4 supplements
The structural basis for haptoglobin-haemoglobin binding by TbHpHbR.
(A) The structure of the complex between T. brucei HpHbR (blue) bound to its ligand, HpSPHb (haptoglobin is yellow, the β-subunit of haemoglobin is red and the α-subunit of haemoglobin is orange). (B…
Figure 2—figure supplement 1
Stereoview of the TbHpHbR in complex with HpHb.
https://doi.org/10.7554/eLife.05553.009
Figure 2—figure supplement 2
Small angle x-ray scattering of complexes of TcHpHbR and TbHpHbR with HpSPHb.
(A) The structure of the TbHpHbR:HpSPHb complex docked into an ab initio molecular envelopes calculated from scattering data. (B) The theoretical scattering calculated from ab initio reconstructions …
Figure 2—figure supplement 3
Clashes between TbHpHbR and a haemoglobin tetramer explain why the receptor does not bind to haemoglobin.
A model for a complex of TbHpHbR bound to haemoglobin. This was derived by docking a haemoglobin tetramer onto the receptor with the β-subunit binding to the receptor as in the TbHpHbR:HpSPHb …
Figure 2—figure supplement 4
The region affected by haptoglobin cleavage is not involved in interaction with TbHpHbR.
(A) The structures of the HpSPHb region of porcine HpHb (red) aligned to the equivalent region of human HpSPHb from the structure of the TbHpHbR:HpSPHb complex (yellow). The structures align with a …
Figure 3
Differences between haptoglobin and haptoglobin-related protein do not alter affinity for TbHpHbR.
(A) The structure of the TbHpHbR:HpSPHb complex is shown with the receptor in blue and haptoglobin in yellow. Side chains in haptoglobin that are different in haptoglobin-related protein are …
Figure 4 with 3 supplements
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 …
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 …
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 …
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 …
Figure 5
A comparison of the dimensions of the TbHpHbR:HpHb complex with those of the N-terminal domains of the variant surface glycoproteins (shown in grey).
This suggests that HpHb will lie at least partially within the VSG layer when bound to two receptors.
Tables
Table 1
Crystallographic data collection statistics
| HpSPHb | Tbb HpHbR | TbbHpHbR:HpSPHb | |
|---|---|---|---|
| Beamline | Diamond I04-1 | Diamond I03 | Diamond I03 |
| Space Group | p3121 | p21 | c2 |
| Cell dimensions (Å) | a = b = 96.6, c = 132.77 | a = 27.90, b = 47.79, c = 203.38, β = 92.79 | a = 223.4, b = 56.59, c = 65.29, β = 92.99 |
| Resolution (Å) | 2.05 | 1.85 | 3.1 |
| Wavelength (Å) | 0.916 | 0.9763 | 0.9750 |
| RPIM (%) | 8.1 (37.4) | 4.5 (42.9) | 6.3 (72.6) |
| I/ σ(I) | 8.7 (2.3) | 10.2 (2.0) | 9.8 (1.6) |
| Completeness (%) | 99.8 (100) | 97.4 (96.5) | 96.9 (97.1) |
| Multiplicity | 9.6 (10.2) | 3.1 (3.1) | 3.2 (3.3) |
Table 2
X-ray refinement statistics
| Complex | HpSPHb | Tbb HpHbR | TbbHpHbR:HpSPHb |
|---|---|---|---|
| Resolution (Å) | 2.05 | 1.85 | 3.1 |
| No. reflections | 43,170 | 44,685 | 17,302 |
| Rwork / Rfree (%) | 18.0 / 22.4 | 19.84 / 23.95 | 19.5 / 21.7 |
| No. of protein residues in model | 544 | 523 | 782 |
| rmsd bond lengths (Å) | 0.020 | 0.017 | 0.012 |
| rmsd bond angles (°) | 2.0 | 1.6 | 1.5 |
| Ramachandran plot | |||
| Allowed region | 89.0% | 98.8% | 92.5% |
| Additional allowed region | 11% | 1.2% | 7.5% |
| Generously allowed region | 0% | 0% | 0% |
| Disallowed region | 0% | 0% | 0% |
Table 3
Small angle x-ray scattering statistics
| MW (kDa) | RG (nm) | Dmax (nm) | Volume (nm3) | Mwapp (kDa) | |
|---|---|---|---|---|---|
| HpSPHb | 59.7 | 2.6 | 7.5 | 75 | 36 |
| TbHpHbR | 32.2 | 3.5 | 11.5 | 44 | 22 |
| TbHbHbR:HpSPHb | 91.8 | 3.2 | 10.8 | 110 | 55 |
| TbHpHbR:HpSPHb | 89.6 | 3.8 | 12.0 | 140 | 70 |
| HpHb | 152 | 5.6 | 18.2 | 214 | 107 |
| TbHpHbR:HpHb | 217 | 6.3 | 16.5 | 370 | 185 |
Table 4
Interactions between TbHpHbR and HpSPHb
| Receptor | HpSPHb | ||||
|---|---|---|---|---|---|
| Residue | Group | Chain | Residue | Group | Interaction |
| Hbβ | |||||
| K56 | side chain | B | Haem144 | O1D | Hydrogen bond |
| E57 | side chain | B | K96 | Side chain | Salt bridge |
| S59 | side chain | B | Haem144 | O1D/O2D | Hydrogen bond |
| I60 | side chain | B | Patch | Hydrophobic | |
| R67 | side chain NH1 | B | R41 | Backbone CO | Hydrogen bond |
| E70 | side chain OE1/OE2 | B | R41 | Side chain NE/NH2 | Salt bridge |
| S161 | side chain | B | K60 | Side chain | Hydrogen bond |
| S161 | side chain | B | S45 | Backbone CO | Hydrogen bond |
| K164 | side chain | B | Haem144 | O2D | Hydrogen bond |
| R199 | side chain NE | B | Haem144 | O2A | Hydrogen bond |
| Y200 | side chain OH | B | Haem144 | O2A | Hydrogen bond |
| S203 | backbone CO | B | K96 | Side chain | Hydrogen bond |
| HpSP | |||||
| S73 | side chain | C | K345 | Side chain | Hydrogen bond |
| V74 | hydrophobic | C | Patch | Hydrophobic | |
| Q75 | OE1 | C | G276 | Backbone CO | Hydrogen bond |
| A78 | side chain | C | Patch | Hydrophobic | |
| A82 | side chain | C | Patch | Hydrophobic | |
| K85 | side chain | C | D305 | Side chain O2D | Salt bridge |
