Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 119, Issue 6, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2113927119
Keywords
membrane transporter; protein dynamics; high-speed atomic force microscopy; single-molecule biophysics
Categories
Funding
- Dutch Research Council (NWO)
- Marie Sklodowska-Curie Actions (MSCA) Individual fellowship [INTERACT 751404]
- MEXT Japan [15K21711]
- European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Actions [860954]
- Grants-in-Aid for Scientific Research [15K21711] Funding Source: KAKEN
- Marie Curie Actions (MSCA) [860954] Funding Source: Marie Curie Actions (MSCA)
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This study used high-speed atomic force microscopy to visualize the transport cycle of the CitS protein in real time. It revealed the presence of three distinguishable states and independent movement of the protein subunits. These findings provide insights into the transport mechanism of the CitS protein.
The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na+ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of similar to 0.5 nm, similar to 1.0 nm, and similar to 1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter GltPh. Since CitS and GltPh are structurally unrelated, we conclude that the three-state elevators have evolved independently.
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