Understanding the mechanisms of multidrug transport proteins

• Applicant: Professor Dr. Lars Schäfer
• Subject Area: Biophysics
• Term: from 2012 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 212212136

• Final Report Year: 2019

Final Report Abstract

The main aim of the Emmy Noether project was to unravel the structure–dynamics–function relationships that underlie the working mechanisms of multidrug transport proteins. To obtain the missing atomic-level insights, molecular dynamics (MD) simulations were carried out, mainly on ABC transporters. ABC transporters are ATP-driven molecular machines in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is coupled to large-scale conformational changes of the transmembrane domains (TMDs) that ultimately translocate substrate molecules across biological membranes. A major outcome of the project was the direct simulation of the large-scale alternating access type conformational transition between an inwardfacing (IF) and an outward-facing (OF) conformation of the ABC exporter TM287/288, a bacterial homolog of the eukaryotic multidrug efflux pump P-glycoprotein. The final OF conformation predicted by the MD simulations, which were unbiased and did not draw on any experimental information other than the initial structure, was later validated by experimental collaborators using EPR spectroscopy and X-ray crystallography. The multi-microsecond MD simulations reveal the full dynamic pathways of the large-scale conformational transition and show how it is orchestrated by tight allosteric coupling between the NBDs and TMDs. In addition to the conformational transition between IF and OF states, the project focused on the chemical mechanism of ATP hydrolysis, another key aspect of the chemomechanical coupling in ABC transporters. Hybrid quantum mechanical/molecular mechanical (QM/MM) free-energy simulations showed that ATP hydrolysis in ABC transporters proceeds in three steps. Interestingly, the overall free energy change associated with the cleavage of the phosphate bond inside the NBD is close to zero, suggesting that phosphate bond cleavage itself cannot provide a power stroke for conformational changes of the transporter. Other transporter related work included the embedding of ABC transporters into lipid nanodiscs and the binding of a substrate to the S-component of an energy coupling factor (ECF) type ABC transporter. Furthermore, the binding of a peripheral membrane protein to a lipid bilayer surface was investigated, with a focus on the hydrationmediated long-range correlations of the atomic motions of the protein and the membrane. Together with additional methodological work on enhanced sampling approaches for biomolecular simulations, the results of the Emmy Noether project pave the way for future studies of membrane transport, including the translocation of substrate molecules.

Publications

• In Vivo Trp-Scanning of the Small Multidrug Resistance Protein EmrE Confirms 3D Structure Models. J. Mol. Biol., 2013, 425, 4642–4651
  P. Lloris-Garcerá, J. S. Slusky, S. Seppälä, M. Prieß, L. Schäfer, G. von Heijne
  
• Mixing Martini: Electrostatic Coupling in Hybrid Atomistic/Coarse-Grained Biomolecular Simulations. J. Phys. Chem. B, 2013, 117, 3516–3530
  T. A. Wassenaar, H. I. Ingolfsson, M. Prieß, S. J. Marrink, L. Schäfer
  
• Substrate-induced Conformational Changes in the S-component ThiT from an Energy Coupling Factor Transporter. Structure, 2013, 21, 861–867
  M. Majsnerowska, I. Hänelt, D. Wunnicke, L. Schäfer, H.-J. Steinhoff, D. J. Slotboom
  
• An Annular Lipid Belt is Essential for Allosteric Coupling and Viral Inhibition of the Antigen Translocation Complex TAP. J. Biol. Chem., 2014, 289, 33098–33108
  S. Eggensperger, O. Fisette, D. Parcej, L. Schäfer, R. Tampé
  
• On Using Atomistic Solvent Layers in Hybrid All-Atom/Coarse-Grained Molecular Dynamics Simulations. J. Chem. Theory Comput., 2015, 11, 4460–4472
  A. B. Kuhn, S. M. Gopal, L. Schäfer
  
• Hydration Dynamics of a Peripheral Membrane Protein. J. Am. Chem. Soc., 2016, 138, 11526–11535
  O. Fisette, C. Päslack, R. Barnes, J. M. Isas, R. Langen, M. Heyden, S. Han, L. Schäfer
  
• Release of Entropic Spring Reveals Conformational Coupling Mechanism in the ABC Transporter BtuCD-F. Biophys. J., 2016, 110, 2407–2418
  M. Prieß, L. Schäfer
  
• Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter. J. Am. Chem. Soc., 2018, 140, 4543–4551
  H. Göddeke, M. H. Timachi, C. A. J. Hutter, L. Galazzo, M. A. Seeger, M. Karttunen, E. Bordignon, L. Schäfer
  
• Molecular Mechanism of ATP Hydrolysis in an ABC Transporter. ACS Cent. Sci., 2018, 4, 1334–1343
  M. Prieß, H. Göddeke, G. Groenhof, L. Schäfer
  
• Atomistic Characterization of Collective Protein- Water-Membrane Dynamics. Phys. Chem. Chem. Phys., 2019
  C. Päslack, L. Schäfer, M. Heyden
  
• On Obtaining Boltzmann-Distributed Configurational Ensembles from Expanded Ensemble Simulations with Fast State Mixing. J. Chem. Theory Comput., 2019, 15, 2774–2779
  S. Wingbermühle, L. Schäfer
  
• The Extracellular Gate Shapes the Energy Profile of an ABC Exporter. Nature Commun., 2019, 10, 2260
  C. A. J. Hutter, M. H. Timachi, L. M. Hürlimann, I. Zimmermann, P. Egloff, H. Göddeke, S. Kucher, S. Stefanic, M. Karttunen, L. Schäfer, E. Bordignon, M. A. Seeger