Structural basis for ligand and innate immunity factor uptake by the trypanosome haptoglobin-haemoglobin receptor

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This manuscript describes the structural investigation of the haptoglobin-haemoglobin receptor (HpHbR) of Trypanosoma brucei in complex with human haptoglobin-haemoglobin(HpHb) by x-ray crystallography as well as SAXS measurements. This is an important interaction in trypanosome molecular biology, as it allows the parasite to acquire haem, and it is relevant to the innate immune response humans have to many species of trypanosome parasites. While the structures of the individual components were largely known previously, the details of their complex were hitherto unknown and are revealed for the first time here. The biological experiments, demonstrating the different efficiencies of receptor-mediated uptake of monomeric and dimeric HpHb ligands are an important component of the paper that nicely supports the structure-based hypotheses on increased ligand avidity made possible by a 'two receptors bind one ligand dimer' model.

Overall, the reviewers consider this a very nice paper showing important results. The reviewers have not found any major concerns but have pointed out some minor issues and questions that are summarized below.

Reviewer #2

1) I would suggest that the authors consider presenting a final figure of how they think a receptor pair/ligand dimer would 'sit' in a sea of VSG molecules (top and side view). This would be helpful for the reader, and I am sure would be popular as a discussion and teaching aid.

2) While I like the receptor pair/ligand dimer model (particularly since it is supported by uptake data) it leaves one quandary not discussed in the paper: how do two HpHbR molecules find each other to bind the ligand dimer with high-avidity? The receptors are GPI anchored (which helps) and so is the VSG coat so they presumably can wander the surface but what is the probability of a 3-way collision (of 2 independent receptors and a ligand dimer) on a 2-dimensional surface when the density of the receptors is low? Is the model of transient single receptor-ligand dimer interactions that are occasionally 'locked down' when another receptor arrives by chance? Some discussion I think is called for (but not additional experiments).

Reviewer #3

I am not an expert on small angle X-ray scattering, but it seems to me that the envelope is a tight fit for the model in Figure 2A and a generous fit in Figure 2B. The authors might comment on this. While these data are largely confirmatory, the SAXS data in Figure 4 are predictive, and there needs to be confidence in the interpretations.

Many of the structure Figures (and especially Figure 2) would benefit from stereo views.

The paragraph “TLF1enters trypanosomes via receptor mediated endocytosis…”, in the Introduction section, is confusing and does not clearly explain what TLF1 and TLF2 are. The nomenclature in this paper (field) is quite complex. I wonder if some sort of introductory figure could help.

The 50 degree kink is interesting and interpreted in terms of sequence variations. It would be useful to add some comment about how main chain hydrogen bonding is affected.

In the Results section (“The structure of TbHpHbR in complex with haptoglobin-haemoglobin”), what does the 1250 Ų refer to? Is it the sum of the areas buried on the partner proteins or is it the interface area?

In the Hb field, the helices have always been denoted by letters, A–H in the case of the β-chain, rather than numbers.

In Table 2, the refinement statistics for TbbHpHbR:HpSPHb look outstanding for a structure at modest resolution especially compared to the Tbb HpHbR structure. The authors should comment briefly on this.

Reviewer #2

1) I would suggest that the authors consider presenting a final figure of how they think a receptor pair/ligand dimer would 'sit' in a sea of VSG molecules (top and side view). This would be helpful for the reader, and I am sure would be popular as a discussion and teaching aid.

We have been hesitant to produce a model showing the structure of the TbHpHbR: HpHb complex in the context of the VSG layer mainly because of our lack of knowledge of the relative sizes of the C-terminal domains of TbHpHbR and VSG. When TbHpHbR adopts a tilted conformation on binding to HpHb it is approximately the same height as a VSG molecule, making it likely that the HpHb will lie, at least partially, within the VSG layer. We have now included a figure to show this similarity, but do not feel comfortable to go further in proposing a detailed model.

2) While I like the receptor pair/ligand dimer model (particularly since it is supported by uptake data) it leaves one quandary not discussed in the paper: how do two HpHbR molecules find each other to bind the ligand dimer with high-avidity? The receptors are GPI anchored (which helps) and so is the VSG coat so they presumably can wander the surface but what is the probability of a 3-way collision (of 2 independent receptors and a ligand dimer) on a 2-dimensional surface when the density of the receptors is low? Is the model of transient single receptor-ligand dimer interactions that are occasionally 'locked down' when another receptor arrives by chance? Some discussion I think is called for (but not additional experiments).

The point made about the likelihood of two receptors colliding with a single HpHb is an interesting one. Assuming that the receptors are predominantly localised at the flagellar pocket, and using the known receptor copy number and VSG diffusion rates, we have calculated that each receptor should contact another receptor approximately once each second. As the t_1/2 for the dissociation of HpHb from a single receptor is in the region of 70-100 s, there is a strong likelihood that an HpHb attached to a single receptor will interact with a second receptor before it dissociates, leading to higher avidity bivalent binding. We have added a paragraph to the Discussion section to make this point.

Reviewer #3

I am not an expert on small angle X-ray scattering, but it seems to me that the envelope is a tight fit for the model in Figure 2A and a generous fit in Figure 2B. The authors might comment on this. While these data are largely confirmatory, the SAXS data in Figure 4 are predictive, and there needs to be confidence in the interpretations.

We agree with the reviewer that it is essential to be confident about the interpretation of the data supporting the formation of a complex containing two receptors bound to a single HpHb in solution. We have made two changes to the manuscript to present data that strengthens this conclusion. Firstly, we now include SAXS data for HpHb alone, allowing comparison with that for the HpHbR:receptor complex. The envelopes derived from these data, together with the accompanying molecular weights, support the binding of two receptors to each HpHb. In addition, we have included SEC-MALLS data, which also shows the formation of a complex containing two receptors bound to a single HpHb. These biophysical data support the conclusions derived from crystallography and explain the in vivo effects observed in HpHb uptake experiments.

Many of the structure Figures (and especially Figure 2) would benefit from stereo views.

We have added a new figure supplement for Figure 2 to show a stereoview of the TbHpHbR:HpSPHb complex.

The paragraph “TLF1enters trypanosomes via receptor mediated endocytosis…”, in the Introduction section, is confusing and does not clearly explain what TLF1 and TLF2 are. The nomenclature in this paper (field) is quite complex. I wonder if some sort of introductory figure could help.

We have clarified the paragraph on the trypanolytic factors in the Introduction, simplifying our description of these (imperfectly characterised) factors and focusing our discussion onto the key ApoLI and Hpr components. We have also rearranged the subsequent two paragraphs. We believe that this will improve their clarity to a reader from outside the field and give a balanced view of what we know of the role of the HpHbR in mediating innate immunity.

The 50 degree kink is interesting and interpreted in terms of sequence variations. It would be useful to add some comment about how main chain hydrogen bonding is affected.

We have added a sentence to the description of the kink in TbHpHbR in which we clarify the effect on main chain hydrogen bonding, and we describe which residues in each of the three helices have their hydrogen bonding disrupted.

In the Results section (“The structure of TbHpHbR in complex with haptoglobin-haemoglobin”), what does the 1250 Ų refer to? Is it the sum of the areas buried on the partner proteins or is it the interface area?

The area of 1250 Ų is the interface area, i.e. the area on the receptor that is covered by HpHb, and not the sum of the buried surface areas on both proteins. We have clarified this in the text.

In the Hb field, the helices have always been denoted by letters, A–H in the case of the β-chain, rather than numbers.

We have corrected the labelling of the haemoglobin β-subunit helices, with helices C and F indicated as those that contact the receptor.

In Table 2, the refinement statistics for TbbHpHbR:HpSPHb look outstanding for a structure at modest resolution especially compared to the Tbb HpHbR structure. The authors should comment briefly on this.

We agree with the reviewer that the refinement statistics for the TbHbHpR:HpSPHb structure are very good. This is, in part, due to the use of additional restraints from the higher resolution structures of the receptor and the HpSPHb complex during refinement. As neither structure showed any conformational change upon complex formation, these additional restraints were used to improve the stereochemistry of the refined model. We were careful, by comparison of the final maps with those obtained without use of these restraints, to show that their use did not lead to major changes in the model. We have increased our explanation of our refinement procedure in the Materials and methods to clarify this.

In addition, we have deposited the pdb codes and diffraction data to the protein data bank with codes 4X0I, 4X0J and 4X0L.