A novel fold for acyltransferase-3 (AT3) proteins provides a framework for transmembrane acyl-group transfer

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Acylation of diverse carbohydrates occurs across all domains of life and can be catalysed by proteins with a membrane bound acyltransferase-3 (AT3) domain (PF01757). In bacteria, these proteins are essential in processes including symbiosis, resistance to viruses and antimicrobials, and biosynthesis of antibiotics, yet their structure and mechanism is largely unknown. In this study, evolutionary co-variance analysis was used to build a computational model of the structure of a bacterial O-antigen modifying acetyltransferase, OafB. The resulting structure exhibited a novel fold for the AT3 domain, which molecular dynamics simulations demonstrated is stable in the membrane. The AT3 domain contains 10 transmembrane helices arranged to form a large cytoplasmic cavity lined by residues known to be essential for function. Further molecular dynamics simulations support a model where the acyl-coA donor spans the membrane through accessing a pore created by movement of an important loop capping the inner cavity, enabling OafB to present the acetyl group close to the likely catalytic resides on the extracytoplasmic surface. Limited but important interactions with the fused SGNH domain in OafB are identified and modelling suggests this domain is mobile and can both accept acyl-groups from the AT3 and then reach beyond the membrane to reach acceptor substrates. Together this new general model of AT3 function provides a framework for the development of inhibitors that could abrogate critical functions of bacterial pathogens.

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  1. Evaluation Summary:

    By integrating a range of computational techniques, the authors generated a structural model for the AT3 domain, which is predicted to adopt a new fold. The key features of the structural model are consistent with the activity of the enzyme as an acyltransferase, with a transmembrane channel that can accommodate an acyl-CoA donor, and an outer cavity formed with a second domain that can accommodate a nascent LPS molecule as substrate. Overall, the study will help stimulate specific experimental analyses that can further evaluate and improve the model for better mechanistic understanding of this class of enzymes. The work will be of interest to structural biologists, and all studying acyltransferase enzymes.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    The authors developed a structural model for the integral membrane AT3 domain and showed that it adopts a novel fold. The structural model is shown to be stable in molecular dynamics simulations and exhibit structural and dynamic features that are consistent with the function of the AT3 domain. The locations of key residues in the structural model are also consistent with functional studies in the literature. The potential binding site of the acetyl-CoA was also evaluated with a quantum mechanical computation, which supported strong binding interactions. The model will play a major role in guiding future experimental studies for targeted mechanistic analyses for this class of important proteins.

  3. Reviewer #2 (Public Review):

    Lipopolysaccharides (LPS molecules) are found on the outer leaflet of the outer membrane in many bacterial species. Composed of non-polar lipid-like acyl tails coupled through a polar headgroup to glycosylations that interact with solution, these molecules are key to forming the outermost barrier critical to bacterial survival. The current study develops a structural model for one of the enzymes in the LPS synthesis pathway. Using co-evolutionary and artificial intelligence methods, the authors develop a structural model for the transmembrane domain that is not in existing structural databases.

    The model is tested with relatively short classical molecular dynamics simulations, on the order of a few hundred nanoseconds. The model is stable over that timeframe, and offers plausible mechanistic hypotheses. There is a putative active site that fits the known activity for the enzyme, and it is possible to fit the reactants into the active site. On its own, this is a highly valuable contribution to the overall literature.

    However, there are elements presented that require further study to fully elucidate. The proposed mechanism for opening and closing two domains within the protein are determined from principal component analysis from simulations that are arguably too short to hope to capture the process fully. The energetic quantification for interactions also needs to be placed into the proper context. While the interaction between an LPS molecule and the protein is substantial when evaluated at the quantum level, this alone tells us nothing about the binding affinity overall, since interactions between LPS and other components within the simulation environment will also be large. Subsequent simulations using free energy techniques such as FEP or umbrella sampling would be required to properly quantify the actual interaction strengths.