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Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	A conserved leucine occupies the empty substrate site of LeuT in the Na+-free return state Lina Malinauskaite , Saida Said , Caglanur Sahin , Julie Grouleff , Azadeh Shahsavar , Henriette Bjerregaard , Pernille Noer , Kasper Severinsen , Thomas Boesen , Birgit Schiøtt , Steffen Sinning , Poul Nissen Nature Communications 7 DOI: 10.1038/ncomms11673 Open Access Show Abstract ▼ Abstract: Bacterial members of the neurotransmitter:sodium symporter (NSS) family perform Na+-dependent amino-acid uptake and extrude H+ in return. Previous NSS structures represent intermediates of Na+/substrate binding or intracellular release, but not the inward-to-outward return transition. Here we report crystal structures of Aquifex aeolicus LeuT in an outward-oriented, Na+- and substrate-free state likely to be H+-occluded. We find a remarkable rotation of the conserved Leu25 into the empty substrate-binding pocket and rearrangements of the empty Na+ sites. Mutational studies of the equivalent Leu99 in the human serotonin transporter show a critical role of this residue on the transport rate. Molecular dynamics simulations show that extracellular Na+ is blocked unless Leu25 is rotated out of the substrate-binding pocket. We propose that Leu25 facilitates the inward-to-outward transition by compensating a Na+- and substrate-free state and acts as the gatekeeper for Na+ binding that prevents leak in inward-outward return transitions.	May 2016
I24-Microfocus Macromolecular Crystallography	Insights into the mechanism of high lipid–detergent crystallization of membrane proteins Sofia Trampari , Caroline Neumann , Samuel J. Hjorth-Jensen , Azadeh Shahsavar , Esben M. Quistgaard , Poul Nissen Journal Of Applied Crystallography 54 DOI: 10.1107/S1600576721010669 Diamond Proposal Number(s): [23316] Show Abstract ▼ Abstract: Obtaining well diffracting crystals of membrane proteins is often challenging, but chances can be improved by crystallizing them in lipidic conditions that mimic their natural membrane environments. One approach is the high lipid–detergent (HiLiDe) method, which works by mixing the target protein with high concentrations of lipid and detergent prior to crystallization. Although this approach is convenient and flexible, understanding the effects of systematically varying lipid/detergent ratios and a characterization of the lipid phases that form during crystallization would be useful. Here, a HiLiDe phase diagram is reported for the model membrane protein MhsT, which tracks the precipitation and crystallization zones as a function of lipid and detergent concentrations, and is augmented with data on crystal sizes and diffraction properties. Additionally, the crystallization of SERCA1a solubilized directly with native lipids is characterized as a function of detergent concentration. Finally, HiLiDe crystallization drops are analysed with transmission electron microscopy, which among other features reveals liposomes, stacked lamellae that may represent crystal precursors, and mature crystals with clearly discernible packing arrangements. The results emphasize the significance of optimizing lipid/detergent ratios over broad ranges and provide insights into the mechanism of HiLiDe crystallization.	Dec 2021
I24-Microfocus Macromolecular Crystallography	The Na+,K+-ATPase in complex with beryllium fluoride mimics an ATPase phosphorylated state Marlene U. Fruergaard , Ingrid Dach , Jacob L. Andersen , Mette Ozol , Azadeh Shahsavar , Esben M. Quistgaard , Hanne Poulsen , Natalya U. Fedosova , Poul Nissen Journal Of Biological Chemistry 298 DOI: 10.1016/j.jbc.2022.102317 Diamond Proposal Number(s): [17844] Open Access Show Abstract ▼ Abstract: The Na+,K+-ATPase generates electrochemical gradients of Na+ and K+ across the plasma membrane via a functional cycle that includes various phosphoenzyme intermediates. However, the structure and function of these intermediates and how metal fluorides mimick them require further investigation. Here, we describe a 4.0 Å resolution crystal structure and functional properties of the pig kidney Na+,K+-ATPase stabilized by the inhibitor beryllium fluoride (denoted E2–BeFx). E2–BeFx is expected to mimic properties of the E2P phosphoenzyme, yet with unknown characteristics of ion and ligand binding. The structure resembles the E2P form obtained by phosphorylation from inorganic phosphate (Pi) and stabilized by cardiotonic steroids, including a low-affinity Mg2+ site near ion binding site II. Our anomalous Fourier analysis of the crystals soaked in Rb+ (a K+ congener) followed by a low-resolution rigid-body refinement (6.9–7.5 Å) revealed preocclusion transitions leading to activation of the dephosphorylation reaction. We show that the Mg2+ location indicates a site of initial K+ recognition and acceptance upon binding to the outward-open E2P state after Na+ release. Furthermore, using binding and activity studies, we find that the BeFx-inhibited enzyme is also able to bind ADP/ATP and Na+. These results relate the E2–BeFx complex to a transient K+- and ADP-sensitive E∗P intermediate of the functional cycle of the Na+,K+-ATPase, prior to E2P.	Sep 2022

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