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ERC

SM-IMPORT Report Summary

Project ID: 638536
Funded under: H2020-EU.1.1.

Periodic Reporting for period 2 - SM-IMPORT (Substrate import at work: single-molecule studies of ABC transporters)

Reporting period: 2017-03-01 to 2017-06-30

Summary of the context and overall objectives of the project

ABC (ATP Binding Cassette) transporters represent the most abundant and diverse family of transport proteins known that play crucial roles in numerous cellular processes. Despite their importance, all proposed molecular models for transport are based on indirect evidence due to the inability of classical biophysical and biochemical techniques to directly visualize dynamic structural changes. To solve this problem, the SM-IMPORT team will decipher the molecular mechanisms of transport using novel single-molecule methodology with the ultimate goal to use this knowledge against pathogenic bacteria, for treatment of ABC-related diseases or multi-drug resistance of cancer cells. The team uses single-molecule fluorescence microscopy for the study of conformational states of an ABC model system in vitro, and thus to observe directly how elementary transport steps are coordinated. This will open up a virtually unexplored biophysical research area and provide a detailed understanding of the molecular mechanisms of ABC transporters.

The overall objectives this proposal are: (i) What is the mechanism of substrate binding in ABC transporters? The conformational equilibrium of ABC-associated substrate-binding proteins will be studied to understand the molecular mechanism of binding. (ii) What are relevant conformational states and changes for substrate translocation? The time- and length-scales of conformational changes in transmembrane and nucleotide binding domains as well as interactions with other domains will be characterized. (iii) How are substrate binding, energy utilization and translocation coordinated in ABC transporters? Finally, a complete model of transport will be developed to decipher the coordination of transport events, i.e., how substrate binding and ATP-hydrolysis are coupled and transferred into conformational changes that drive substrate transport.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Work during the first 18 months (period I):
(aim i): Seven different SBDs (SBD1, SBD2, OpuAC, CynR, MalE, FeuA, OppA) were analysed with smFRET in solution and dynamically on the surface. The results showed their binding mechanisms to be induced fit (except for new protein CynR). The SM-IMPORT team also used the quenching properties of the FeuA-ligand ferri-bacilibactin to understand that the ligand binds the open state of the protein. The team published results on improved fluorophores usable in smFRET experiments using an ABC-importer SBD as a model system in Nature Communications. The team is currently preparing publications related to aim 1/2 of the action on (i) structural evolution of ligand-binding mechanisms, (ii) impact of conformational states dynamics on transport in ABC importers, (iii) characterization and relevance of intrinsic conformational changes in SBDs.

(aim ii): The team generated an improved homology model of the OpuA structure to allow design of the smFRET experiments. Subsequently, 27 OpuA-TMD cysteine mutants were designed and made for smFRET analysis. The mutants were screened for ATPase activity and their fluorophore labelling efficiency; mutants with high efficiency and activity were selected for further analysis. The team currently uses wildtype OpuA and a periplasmic (internal plasmid name p178) and cytoplasmic mutant (internal plasmid name p163) for smFRET experiments and functional assays. Overall many fundamental hurdles have been taken, including the labelling of reconstituted OpuA in nanodiscus, but the quality of the data from OpuA in smFRET experiments has to be improved to generate publishable data. The team further studied the ligand-dependence of conformational dynamics of various proteins (SBD1/2, OpuAC, MalE) and the relevance of the specific conformational states for ABC importers; these results reveal novel mechanisms in SBD-TMD interactions for ABC importers; a paper is in preparation. Cordes and two team members have further established a collaborative project and realized a smFRET study of protein with nucleotide binding domains similar to the ones in ABC importers; the results of this model system ABCE1, a ribosome-splitting factor, are currently summarized and prepared for publication.

(aim iii): In this project part, mostly technical advances have been made and most tasks in the aim are planned for upcoming periods.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Especially in (aim i) the group has made substantial progress beyond planned activities and succeeded in understanding protein structure-function relationships and the evoluationary aspects of how proteins diversify their function. The results are currently prepared for publication. The generality of the found results will likely allow to transform the basic principles established here into guidelines for drug design in the direction of antibiotics.
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