Post by Mattia Sturlese, PhD.
Recently, we derived the structure of the prestin transmemebrane domain by combining bioinformatics, homology modeling, and MD simulations with extensive experimental work. Prestin (SLC26A5), a member of the SLC26/SulP anion transporter family localized in the outer plasma membrane of the outer hair cells (OHCs), is a motor protein essential for auditory processing. Motor proteins are a fascinating class of proteins able to transform molecular events into movements.
Prestin, a highly responsive piezoelectric transducer of unclear mechanism of action
Classical molecular motors are driven by ATP hydrolysis while prestin acts through a chlorine dependent intrinsic voltage sensor (IVS). In prestin, intracellular binding of a chloride ion induces a very large structural rearrangement – determined by the membrane potential – that endows this motor protein with a motility-related charge movement or nonlinear capacitance (NLC). One of the most attractive peculiarities of this mechanism relies on time scale in which this molecular event occurs, as it is remarkably faster than that of other cellular motor proteins. In a nutshell, prestin is a piezoelectric transducer able to convert membrane potential into macromolecular movement extremely efficiently. In fact, the piezoelectric coefficient in OHCs (20 fC/nN) is four orders of magnitude greater than found in man-made crystals used in electronic devices or manipulators. Despite its established physiological role as a key element in the high acuity of mammal hearing through cochlear amplification, it is essentially unknown how prestin can generate mechanical force. To date there are no known structures for the SLC26/SulP superfamily of anion transporters.
Computational modeling towards a predicted 3D prestin structure
Sequence-based bioinformatic analyses indicated the NCS2 and SulP transported families as possible candidates for homology modeling. Using hidden Markov models (HMMs) and their profile analysis we identified the uracil transporter UraA (PDB: 3QE7) as the best and most meaningful template for homology modeling. In order to validate the biophysics of this model we collected 150ns of atomistic molecular dynamics simulations using ACEMD. The MD simulation data showed that although there were defined local differences between the two models the two conserved the global fold and topology of UraA at equilibrium, thus corroborating the validity of our model. Thus, the prestin transmembrane domain appeared to be composed of 14 transmembrane segments organized into a 7+7 inverted repeat fold. Appropriate 3D structures to be used in further analysis were obtained by cluster selection.
Experimental validation of the model and understanding of the molecular basis for prestin’s activity
We continued validating the topology of the homology model using a substituted Cys accessibility method (SCAM).The SCAM results showed a notable agreement with water-protein contacts as derived from MD simulations. Importantly, the pattern of accessibility is in full agreement with the predicted topology (Fig 1), corroborating the model and UraA as an appropriate template for modelling SLC26. Solute accessible pathways were identified by analyzing the internal solvated areas and calculating the residence probability of explicit water molecules during the last 50 ns of the MD simulation. The central cavity is readily accessible from the intracellular space but occluded from the extracellular space by a constriction that precludes permeation from the exterior to the central cavity. A SCAM scan along the entire TM10 provided experimental confirmation of the model results; amino-acid positions predicted to contribute to the central cavity and its intracellular entrance were accessible to intracellular, but not extracellular MTS reagents. The accuracy of the model and the information from the MD simulations has also allowed identification of a candidate central binding site validated experimentally though mutagenesis.
Reference and further reading:
*Gorbunov, D., *Sturlese, M., Nies, F., Kluge, M., Bellanda, M., Battistutta, R., and ^Oliver, D. (2014). Molecular architecture and the structural basis for anion interaction in prestin and SLC26 transporters. Nat Commun 5.
Dallos, P., and Fakler, B. (2002). Prestin, a new type of motor protein. Nat Rev Mol Cell Biol 3, 104–111.
Oliver, D., He, D.Z.Z., Klöcker, N., Ludwig, J., Schulte, U., Waldegger, S., Ruppersberg, J.P., Dallos, P., and Fakler, B. (2001). Intracellular Anions as the Voltage Sensor of Prestin, the Outer Hair Cell Motor Protein. Science 292, 2340 –2343.
Zheng, J., Shen, W., He, D.Z.Z., Long, K.B., Madison, L.D., and Dallos, P. (2000). Prestin is the motor protein of cochlear outer hair cells. Nature 405, 149–155.