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Structural and energetic analysis of metastable intermediate states in the E1P-E2P transition of Ca²⁺-ATPase.

ABSTRACT: Sarcoplasmic reticulum (SR) Ca²⁺-ATPase transports two Ca²⁺ ions from the cytoplasm to the SR lumen against a large concentration gradient. X-ray crystallography has revealed the atomic structures of the protein before and after the dissociation of Ca²⁺, while biochemical studies have suggested the existence of intermediate states in the transition between E1P⋅ADP⋅2Ca²⁺ and E2P. Here, we explore the pathway and free energy profile of the transition using atomistic molecular dynamics simulations with the mean-force string method and umbrella sampling. The simulations suggest that a series of structural changes accompany the ordered dissociation of ADP, the A-domain rotation, and the rearrangement of the transmembrane (TM) helices. The luminal gate then opens to release Ca²⁺ ions toward the SR lumen. Intermediate structures on the pathway are stabilized by transient sidechain interactions between the A- and P-domains. Lipid molecules between TM helices play a key role in the stabilization. Free energy profiles of the transition assuming different protonation states suggest rapid exchanges between Ca²⁺ ions and protons when the Ca²⁺ ions are released toward the SR lumen.

SUBMITTER: Kobayashi C

PROVIDER: S-EPMC8501872 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ATP Binding Cassette (ABC) transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs), the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg(2+) with each NBD indicates that the coordination of ATP and Mg(2+) differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation of the simulations.

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Structural and energetic analysis of metastable intermediate states in the E1P-E2P transition of Ca²⁺-ATPase.

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Structural and energetic analysis of metastable intermediate states in the E1P-E2P transition of Ca2+-ATPase.

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Structural and energetic analysis of metastable intermediate states in the E1P-E2P transition of Ca²⁺-ATPase.