Dynamics of P-type ATPase transport revealed by single-molecule FRET

Abstract

P-type ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest a transport mechanism defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have employed single-molecule fluorescence resonance energy transfer (smFRET) to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. Using the smFRET approach we identify key intermediates with no known crystal structures, and our findings delineate reversible and an essentially irreversible step in the transport process wherein Ca2+ efflux by LMCA1 is rate limited by phosphoenzyme formation and resolved by ADP and Ca2+ release leading to an open E2P state.

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

“…In recent years, it has, however, become apparent that transporters show conformational dynamics and that those have to be understood and characterized (Locher, 2016). For this, single‐molecule Förster resonance energy transfer (smFRET; Lerner et al , 2018) has been used to monitor these conformational dynamics of transporters, e.g., the P‐type Ca 2+ ‐ATPase (LMCA1) from Listeria monocytogenes (Dyla et al , 2017), secondary transporters such as the aspartate/Na + symporter from Pyrococcus horikoshii (Akyuz et al , 2013), and the leucine/Na + symporter LeuT from Aquifex aeolicus (Zhao et al , 2010). While there are also smFRET studies of ABC importers regarding the conformational dynamics of the substrate binding domains (SBDs; Gouridis et al , 2015) or interactions of SBD‐TMD (Goudsmits et al , 2017a; Yang et al , 2018), there are no studies of conformational dynamics and crosstalk between TMDs and NBDs in ABC exporters in native‐like lipid environment (Verhalen et al , 2012).…”

Multivalent Chelators for In Vivo Protein Labeling

“…[28] Thes tudy of conformational dynamics via smFRET was further realized by as imultaneous,f our-color single-molecule fluorescence methodology. [30] Remarkably, the trisNTA-His-tag interaction enabled the highly specific, reversible,a nd oriented attachment of LMCA1. Another field of application in smFRET is the utilization of trisNTAf or the site-specific,o riented, and high-affinity attachment of His-tagged proteins on surfaces.F or instance, Listeria monocytogenes Ca 2+ -ATPase (LMCA1), aPhosphorylation-type (P-type) ATPase,w as anchored to as parsely trisNTA-decorated interface to observe conformational changes associated with functional transitions.…”

The crystal structure of the Ca2+-ATPase 1 fromListeria monocytogenesreveals a pump primed for dephosphorylation

“…smFRET experiments have shown that the pump dephosphorylates rapidly and indicate that the required interactions form quickly [11]. This suggests that with fewer interactions involved, a catalytically competent state is reached faster in LMCA1.…”

“…The structure was determined and partially refined at 4.3 Å resolution [21], but did not permit any detailed analysis. We have also investigated LMCA1 using single-molecule FRET (smFRET) [11,22].…”