"Crystallography had allowed the identification of only one structural form of the molecule, but our experiments and computations were able to identify how this form changes and thereby add an understanding of the functional role of the different forms that the molecule must adopt to accomplish transport activity," says Dr. Javitch.
The main surprise was the realization that two binding sites on the transporter molecule need to be filled simultaneously and cooperate in order for transport to be driven across the cell membrane. For these studies, the scientists used the crystal structure of a bacterial transporter that is very similar to human neurotransmitter transporters. They performed computer simulations to reveal the path of the transported molecules into cells. Laboratory experimentation was used to test the computational predictions and validate the researchers' inferences.
Together, these procedures revealed a finely-tuned process in which two sodium ions bind and stabilize the transporter molecule for the correct positioning of the two messenger molecules -- one deep in the center of the protein, and the other closer to the entrance. Like a key engaging a lock mechanism, this second binding causes changes in the transporter throughout the structure, allowing one of the two sodium molecules to move inward, and then release the deeply bound messenger and its sodium partner into the cell.
In the bacterial transporter studied, antidepressant molecules bind in the outer one of two sites, and stop the transport mechanism, leaving the messenger molecule outside the cell.
The second team of researchers, involving a collaboration of the Weinstein and Javitch labs with colleagues in Denmark (the labs of U
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New York- Presbyterian Hospital/Weill Cornell Medical Center/Weill Cornell Medical College