Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Reviewer #1 (Public Review):

Summary:
TRAP transporters are an unusual class of secondary active transporters that utilize periplasmic binding proteins to deliver their substrates. This paper contributes a new 3 Å structure of the Haemophilus influenzae TRAP transporter. The structure joins two other recent cryo-EM structures of TRAP transporters, including a lower-resolution structure of the same H. influenzae protein (overall 4.7 Å), and a ~3 Å structure of a homologue from P. profundum. In addition to reporting a higher resolution cryo-EM structure, the authors also recapitulate protein activity in a reconstituted system, investigate protein oligomerization using analytic ultracentrifugation, and evaluate interactions and function in "mix and match" configurations with periplasmic subunits from other homologues.

Strengths:
The strength of the paper is that the better resolution cryo-EM data permits sidechain assignment, the identification of bound lipids, and the identification of sodium ions. It is important to get this structure out there since the resolution passes an important threshold for model-building accuracy. The current structure nicely explains a lot of prior mutagenesis data on the H. influenzae TRAP. This is also the first structure of a TRAP protein to be solved without a fiducial, although the overall structure is not very different from those solved with fiducials.

Weaknesses:
The experiments examining the monomer/dimer equilibrium appear somewhat preliminary. The biological or mechanistic importance of oligomerization is not established, so these experiments are inherently of limited scope. Moreover, cryo-EM datasets exhibit both parallel and antiparallel dimers, the latter of which are clearly not biologically relevant. It is probably impossible to distinguish these in the AUC experiments, which makes interpretation of these experiments more difficult.

Similarly, the importance of the lipid binding sites observed in cryo-EM isn't experimentally established (for example by mutating the binding site) and it thus seems too preliminary to infer that they are important for function.

Reviewer #2 (Public Review):

Summary:
In this manuscript, the membrane component of the sialic acid-specific TRAP transporter, SiaQM (HiSiaQM), from H. influenzae, is structurally characterized. TRAP transporters are substrate binding protein (SBP)-dependent secondary-active transporters, and HiSiaQM is the most comprehensively studied member of this family. While all previous work on fused TRAP transporter membrane proteins suggests that they are monomeric (including the previous structural characterization of HiSiaQM by a different group), a surprising finding from this work is the observation that HiSiaQM can form higher oligomers, consistent with it being a dimer. These higher oligomeric states were initially observed after extraction of the protein with LMNG detergent but were also observed in DDM detergent, amphipol and nanodiscs using analytical ultracentrifugation (AUC). Structural characterization of dimeric HiSiaQM revealed 2 arrangements, parallel and antiparallel arrangements, the latter of which is unlikely to be physiologically relevant.

The higher resolution of this new structure of HiSiaQM (2.2-2.7 Å compared to 4.7 Å for the previous structure) facilitated the assignment of bound lipids at the dimer interface and a lipid molecule embedded in each of the protomers; allowed for a clearer refinement of the Na+ and putative substrate binding sites, which differ slightly from the previous structure; and produced better-modelled side chains for the residues involved in the SBP:HiSiaQM interaction. The authors developed a useful AUC-based assay to determine the affinity for this interaction revealing an affinity of 65 µM. Finally, the authors make the very interesting observation that a sialic acid-specific SBP from a different TRAP transporter can utilize HiSiaQM for transport, contrary to previous observations, revealing for the first time that TRAP membrane components can recognize multiple SBPs.

Overall, this is a well-written and presented manuscript detailing some interesting new observations about this interesting protein family. One of the main findings, that the protein can form a dimer, is supported by data, but the physiological relevance of this is questionable, and the possibility that this is artefactual has not been ruled out. Conclusions regarding the mechanistic importance of the lipid-binding sites are not currently supported by the data.

Strengths:
The main strength of this work is the increased resolution of HiSiaQM, which allows for a much more precise assignment of side chains and their orientation. This will be of importance for subsequent mechanistic studies on the contributions of these residues to Na+ and sialic acid binding and conformational changes.

The observation of the lipids, especially the lipid embedded near the fusion helix, is an intriguing observation, which lays the groundwork for future work to understand the lipid-dependence of these transporters. The development of the AUC-based approach to measure SBP affinity for the membrane component will likely prove useful to future studies.

Weaknesses:
One of the main results from this work is the observation that HiSiaQM can form a dimer. Two arrangements were observed, parallel and antiparallel, the latter of which is almost certainly physiologically irrelevant as it would preclude essential interactions with the extracytoplasmic substrate-binding protein. As acknowledged by the author, this non-physiological arrangement is likely a consequence of protein preparation (overexpression, extraction, purification, etc.). However, if one dismisses the antiparallel arrangement as non-relevant and an artefact of protein preparation, it is difficult for the parallel arrangement to maintain its credibility, as it was also processed in the same way. This is especially true when one considers that there is only 100 Å2 buried surface area in the parallel arrangement that does not involve any sidechains; it is difficult to envisage this as a specific interaction, e.g. compared to related proteins that have ~2000 Å2 buried surface area. Unless this dimerization is observed in a bacterial membrane at physiological protein concentrations, it is difficult to rule out the possibility that the observed dimerization is merely an artefact caused by the expression, purification and concentration of the protein.

The manuscript contains some excellent structural analysis of this protein, whose higher resolution reveals some new and interesting insights. However, a weakness of the current work is a lack of validation of these observations using other approaches. For example, lipid interactions are observed in the structure that the authors claim is mechanistically important. However, without disrupting these interactions to look at the effect on transport, this conclusion is not supported. Similarly, the authors use their structure to predict residues that are important for the SBP:membrane protein interaction, and they develop an AUC-based binding assay to study this interaction, but they do not test their predictions using this approach.

Reviewer #3 (Public Review):

Summary:
The manuscript reports new molecular characterization of the Haemophilus influenza tripartite ATP-independent periplasmic (TRAP) transporter of N-acetylneuraminate (Neu5Ac). This membrane transporter is important for the virulence of the pathogen. H. influenza lacks Neu5Ac biosynthetic pathway and utilizes the TRAP transporter to import it. Neu5Ac is used as a nutrient source but also as a protection from the human immune response. The transporter is composed of two fused membrane subunits, HiSiaQM, and one soluble, periplasmic subunit HiSiaP. HiSiaP, by binding to the substrate Neu5Ac, changes its conformation, allowing its binding to HiSiaQM, followed by Neu5Ac and Na+ transport to the cytoplasm. The combination of structural, biophysical and biochemical approaches provides a solid basis for describing the functioning of the Haemophilus influenza Neu5Ac TRAP transporter, which is essential for the pathogen virulence.

Strengths:
The paper describes the electron microscopy structure of HiSiaQM, thanks to its solubilization in L-MNG followed by the exchange to amphipol or nanodisc. In these conditions, HiSiaQM consists of a mixture of monomers and dimers, as characterized by analytical ultracentrifugation. The cryo-EM analysis shows two types of dimers: one in an antiparallel configuration, which is artifactual, and a parallel one, which may be physiologically relevant. Cryo-EM on the dimers allows high-resolution (≈ 3 Å) structure determination. The structure is the first one of a fused SiaQM, and is the first obtained without megabody. The work highlights structural elements (fusion helix, lipids) that could modulate transport. The authors checked the functionality of the purified HiSiaQM, which, after reconstitution in liposome, displays a significantly larger Neu5Ac transport activity compared to the non-fused PpSiaQM homolog. The work identifies Na+ binding sites, and the putative Neu5Ac binding site. From analytical ultracentrifugation using fluorescently labelled HiSiaP, the authors show that HiSiaP is able to interact with HiSiaQM monomer and dimer, with a low but physiologically relevant affinity. HiSiaP interaction with HiSiaQM was modelled using AlphaFold2, and discussed in view of published activity on mutants, and new transport activity assays using SiaQM and SiaP from different organisms. In conclusion, the combination of structural, biophysical and biochemical approaches provides a solid basis for describing the functioning of this TRAP fused transporter.

Weakness:
This work evidences in vitro a HiSiaQM dimer, whose in vivo relevance is not ascertained. However, the authors are very careful, not to over-interpret their data, and their conclusions regarding the transporter structure and function are valid irrespective of its state of association.