Cryo‐EM structure of MsbA in saposin‐lipid nanoparticles (Salipro) provides insights into nucleotide coordination

Kehlenbeck, Dominique-Maurice; Traore, Daouda A K; Josts, Inokentijs; Sander, Simon; Moulin, Martine; Haertlein, Michael; Prévost, Sylvain; Forsyth, V. T.; Tidow, Henning

doi:10.1111/febs.16327

Cited by 16 publications

(12 citation statements)

References 60 publications

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“…15 – 16 , Supplementary Table 8 , and Supplementary Note 2 ). The structure is similar to that of the vanadate-trapped MsbA from S. typhimurium but differs from the previously reported vanadate-trapped, occluded MsbA structures 17 , 47 , largely in the TMD (Supplementary Fig. 17 ).…”

Section: Resultssupporting

confidence: 63%

Structural basis for lipid and copper regulation of the ABC transporter MsbA

et al. 2022

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A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although LPS binding to the inner cavity of MsbA is well established, the selectivity of MsbA-lipid interactions at other site(s) remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5’-diphosphate and vanadate, revealing a distinct KDL binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KDL binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis.

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Section: Resultssupporting

confidence: 63%

Structural basis for lipid and copper regulation of the ABC transporter MsbA

et al. 2022

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“…Likewise, the same stoichiometry of trapping was reported for other ABC transporters in the presence of vanadate, such as the P-glycoprotein, LmrA, or the maltose transporter . In contrast, the 3D structures of the maltose transporter and MsbA solved in the presence of ATP:Vi strongly support the presence of two ADP:Vi bound per transporter . It was argued that this difference in stoichiometry could be due to a nonequilibrium process in the biochemical experiments where the free nucleotides (or analogues) had to be removed from the sample.…”

Section: Discussionsupporting

confidence: 52%

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA

et al. 2022

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The detailed mechanism of ATP hydrolysis in ATPbinding cassette (ABC) transporters is still not fully understood. Here, we employed 31 P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The 31 P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg 2+ :vanadate, while the second site is more 'open' allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric 31 P− 31 P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the 13 C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.

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“…S8). The structure is similar to that of the vanadate-trapped MsbA from S. typhimurium but differs from the previously reported vanadate-trapped, occluded MsbA structures, 17,45 Probing the exterior KLA binding sites. A series of MsbA mutants engineered to impact KLA binding were evaluated.…”

Section: X-ray Structure Of the N-terminus Of Msba Coordinated To Cop...supporting

confidence: 46%

Structural basis for lipid and copper regulation of the ABC transporter MsbA

Lyu

Liu

Zhang

et al. 2022

Preprint

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A critical step in Lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although MsbA has been extensively studied, the selectivity of MsbA-lipid interactions remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KLA). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5’-diphosphate and vanadate, revealing an unprecedented KLA binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KLA binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis.

show abstract

Cryo‐EM structure of MsbA in saposin‐lipid nanoparticles (Salipro) provides insights into nucleotide coordination

Cited by 16 publications

References 60 publications

Structural basis for lipid and copper regulation of the ABC transporter MsbA

Structural basis for lipid and copper regulation of the ABC transporter MsbA

Solid-State NMR Reveals Asymmetric ATP Hydrolysis in the Multidrug ABC Transporter BmrA

Structural basis for lipid and copper regulation of the ABC transporter MsbA

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