Conformational dynamics of an ABC exporter investigated by NMR spectroscopy and PET fluorescence quenching

• Applicants: Professorin Dr. Ute Hellmich; Privatdozent Dr. Hannes Neuweiler
• Subject Area: Structural Biology ; Biochemistry ; Biophysics
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 421388231

ATP binding cassette (ABC) transporters are trans-membrane protein assemblies that accomplish the active transport of various molecular compounds, such as small organic molecules, sugars, peptides or ions, across the membrane of living cells. ABC transporters are of medical relevance because their ATPase-driven transport mechanism can lead to drug resistance and transporter dysfunctions can cause severe diseases. The molecular details of the functional mechanism of ABC transporters, however, are only partially understood. The very nature and sequence of conformational changes, which are triggered by ATP binding and hydrolysis at the so-called nucleotide binding domains (NBDs) and lead to transport of a substrate through the transmembrane domains (TMDs), are currently unclear. Pathways of allosteric communication remain to be explored. Here, we combine NMR spectroscopy with fluorescence quenching by photoinduced electron transfer (PET), two complementary high-resolution tools in protein dynamics, to gain new insights into the functional mechanism of the LmrA ABC exporter from L. lactis. While NMR spectroscopy yields atomistic information on protein conformation and dynamics, PET fluorescence spectroscopy explores correlated motions across local or remote structural elements and delivers time constants of conformational change. We start our investigation on the highly-conserved NBD in isolation, where we map out the dynamic consequences of nucleotide binding by NMR, including relaxation-dispersion and 1H/2H exchange measurements, and probe reconfiguration time constants of distinct structural elements with nanosecond time resolution using PET in combination with fluorescence correlation spectroscopy (PET-FCS). Next, we will determine the consequences of interactions with the trans-membrane domain (TMD) on the dynamics of the NBD using 19F NMR experiments and PET-FCS of the NBD in context of the full-length ABC exporter. Finally, we will probe dimerization of NBDs triggered by nucleotide binding and the global tilting motions of TMD helices that translocate a ligand using a tailored PET fluorescence reporter design. We anticipate that the results of our project, where NMR and PET fluorescence spectroscopy as two complementary high-resolution solution techniques are combined for the first time, provide new insights into the functional dynamics of ABC transporters and thereby enhance our understanding of the structural and dynamical basis of substrate transport in this important class of membrane proteins.

• DFG Programme: Research Grants