Scientists at the US Department of Energy’s (DOE’s) Brookhaven National Laboratory have determined the atomic-level structure of zinc transporter protein, the molecular machine that regulates the levels of these essential micronutrients inside the cell. As described in a paper just published in Nature Communications, the structure shows how a cell membrane protein changes shape to move zinc from the environment into the cell and automatically temporarily blocks this activity when zinc levels inside the cell become too high.
“Zinc is essential for many biological activities, but too much can be a problem,” said Kun Liu, a biophysicist at Brookhaven Laboratory who led the project. “In the course of evolution, different organisms have developed many ways to regulate zinc. But no one has proven that a transporter that controls the absorption of zinc from the environment can regulate its own activity. Our study is the first to demonstrate such a zinc transporter. A built-in sensor.”
The research was conducted as part of Brookhaven Lab’s Quantitative Plant Sciences Initiative (QPSI). Using a bacterial version of a zinc transporter that shares important features with zinc transporters in plants, scientists have gained key insights into how these proteins work.
“This research is part of our efforts to understand how plants take up micronutrients like zinc, so we can understand how to design plants that grow at the extremes to produce bioenergy,” said John, head of biology at Brookhaven Lab. Shanklin, co-author on the paper.
The research may also suggest ways to develop food crops with higher zinc content to improve their nutritional value, the scientists noted.
Cryo-EM plus computation
To solve the protein’s structure, the Brookhaven team used cryo-electron microscopy (cryo-EM) at the Laboratory for Biomolecular Structures (LBMS). With this technique, scientists can sample many different conformations of a protein instead of a single crystallized form. This is important because in nature proteins are dynamic rather than static; their parts move.
Cryo-EM does not require proteins to form crystals, so we can take dynamic steps that cannot be done with X-ray crystallography, another method of studying protein structures. Basically, with cryo-EM, we can capture more frames of the ‘movie’ to obtain a structure that is very useful for understanding the biological function of a protein.”
Kun Liu, a biophysicist at Brookhaven Lab
Scientists need powerful computing tools to sort out the many changes in structure. These include artificial intelligence approaches that use machine learning, some of which Liu developed. Using these algorithms, scientists can semi-automatically select and sort millions of cryo-EM images to find groups of similar structures. The method allows them to achieve the highest possible resolution, thereby revealing atomic-scale details of the structure.
For this study, this cryo-EM approach revealed key features of one step of the ZIP (Zrt-/Irt-like protein) zinc transporter, revealing how it regulates its zinc uptake activity depending on how much zinc is available in the cell. .
“Our new data have forced us to reconsider previous views on how this protein works,” Liu said.
Tilt to enter, feel to stop
An earlier report based on X-ray crystallography and coevolutionary analyzes suggested that the carrier may function as an “elevator” for zinc transport. A new study shows how zinc interactions on both sides of the cell membrane move protein parts to bring zinc into the cell and, crucially, block its entry when levels inside get too high.
“Our basic premise is that when the level of zinc inside the cell rises to a certain level – above the level needed to meet the needs of the cell; shows that excess zinc binds to a loop on the inside of the membrane,” Liu said. “Then, when this flexible loop is reoriented, it folds back on itself and binds in a way that blocks zinc from entering the cell.”
“It looks like the plug will go down the bathroom drain and clog it,” Shanklin added.
The scientists also discovered how other parts of the protein move to allow zinc to enter.
When the zinc level inside the cell is low, the zinc will drop out of the loop section and back out of the plug carrier. Zinc in the environment can transfer to the carrier. Inside the transporter, zinc moves part of the protein machine up and tilts, closing the exit to the outside environment. Once the zinc enters the cell, the device resets itself to work again.
“Our cryo-EM structure is the first to show how this loop domain of the protein modulates transporter activity via zinc level-dependent feedback,” Liu said.
This is also known as a dimer, the first structure to show that this zinc transporter is an arrangement of two identical proteins. “It takes two molecules to do the job,” Liu said.
The scientists believe that the presence of two molecules acting as a dimer may have implications for its function or stability – and they will study how the molecules work together with future computational simulations.
“This study provides new methods of zinc transporters in microbes and plants to optimize their growth in areas where zinc is too low or too high, limiting the production of bioenergy and bioproducts,” said Liu.
This research was funded by the DOE Office of Science, Office of Biological and Environmental Research (BER) through QPSI, with support from the Office of Protein Expression, Purification, and Basic Energy Sciences (BES) and sample preparation. LBMS operations are supported by BER.
DOE/Brookhaven National Laboratory
Pang, S., etc. (2023). Structural mechanism of intracellular autoregulation of zinc uptake in ZIP transporters. Nature communications. doi.org/10.1038/s41467-023-39010-6.
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