Organisms must take up nutrients and extrude toxins and waste to survive, grow, and reproduce. These import and export functions are performed at cellular membranes by proteins referred to as transporters. The thousands and thousands of transporters found in nature take on many different 3D folds (think protein origami), although a few folds are very common, suggesting that these folds arose early in evolution and are particularly well suited to the transport task. LeuT-fold transporters (named after the bacterial amino acid transporter LeuT) constitute one of the largest superfamilies of transporters. Members of this superfamily are either importers, exporters, or exchangers, and they transport a wide variety of substrates, including transition metal ions, potassium or chloride ions, neurotransmitters, amino acids, and sugars.
Substrate transport requires that transporters undergo internal motions, or conformational changes. With hundreds of accumulated experimental 3D structures of LeuT-fold transporters, researchers have anecdotally noted similarities and differences in their conformational changes. In our lab, which has deeply investigated the conformational changes of one such LeuT-fold transporter, discussions frequently arose around the question: To what extent are variations of conformational changes supported by the LeuT fold?
When research turned virtual in March 2020, Jacob Licht (who had just joined the Gaudet lab as an undergraduate) and Michael Gutierrez (then a post-baccalaureate in the Gaudet lab) needed a new, computational project, and decided to systematically analyze the conformational changes of LeuT-fold transporters. They first gathered and catalogued ~300 LeuT-fold transporter structures. They then devised tailored distance difference matrices to quantitatively compare conformational changes. An advantage of distance difference matrices is that they rely solely on the internal coordinates of each structure and thus avoid a potential perspective bias that can be introduced by a structural alignment, which requires choosing which structural elements to use for the alignment (see the video).
Jacob then went on to complete the systematic comparisons as his undergraduate senior research thesis. We then enlisted the help of Sam Berry, a graduate student in the lab, who performed statistical analyses to identify the recurring conformational change motifs — transporter dance moves, essentially — and quantitatively describe the choreography that each transporter uses.
In a new publication, we present the resulting dataset and analysis, and we make important observations that clarify several long-standing questions:
Although previous reports have questioned whether this is the case, we find that all LeuT-fold transporters we investigated share a common motion known as the “bundle-hash rock,” in which two sets of four transmembrane helices — the bundle and the hash, respectively in yellow and blue in the video — rock relative to each other.
We identify five additional repeatedly observed conformational movements (dance moves), that are layered on the common bundle-hash rock to provide different ways of opening and closing different LeuT-fold transporters to transport substrates across cell membranes.
Although the bundle-hash rock is a common motion, whether the bundle or the hash moves relative to the plane of the cellular membrane differs between LeuT-fold transporters.
Overall, our analysis demonstrates the utility of systematic structural comparisons in understanding the functional mechanisms of membrane transporters. It also suggests that similar studies could provide deeper insights into the conformational dynamics of other protein families. We are in the midst of extraordinary growth in information on protein structure and dynamics both through powerful experimental techniques like cryogenic electron microscopy and computational tools like molecular dynamics simulations and artificial intelligence-based tools like AlphaFold. The resulting deep datasets are ripe for systematic quantitative analyses like the ones we have developed to learn more about the dance styles and dance vocabulary of all sorts of proteins.
The video shows approximations of the conformational changes of the LeuT transporter based on experimental structures. The bundle helices are yellow, the hash helices blue, arms in green, and extra helices in grey. The cellular membrane would form a horizontal plane across the screen. Three possible cases are illustrated:
Left: An overall structure-based alignment suggests all elements are moving relative to each other.
Middle: A hash-based alignment shows the hash as rigid and still as the bundle rocks relative to the hash.
Right: A bundle-based alignment shows the bundle as mostly rigid and the hash rocking relative to the bundle.
Our systematic analyses determined that the middle panel is a more accurate representation of the LeuT dance in the plane of the cell membrane.
by Rachelle Gaudet
