Structures of LeuT in bicelles define conformation and substrate binding in a membrane-like context

Abstract

Neurotransmitter sodium symporters (NSSs) catalyze the uptake of neurotransmitters into cells, terminating neurotransmission at chemical synapses. Consistent with the role of NSSs in the central nervous system, they are implicated in multiple diseases and disorders. LeuT, from Aquifex aeolicus, is a prokaryotic ortholog of the NSS family and has contributed to our understanding of the structure, mechanism and pharmacology of NSSs. At present, however, the functional state of LeuT in crystals grown in the presence of n-octyl-β-D-glucopyranoside (β-OG) and the number of substrate binding sites are controversial issues. Here we present crystal structures of LeuT grown in DMPC-CHAPSO bicelles and demonstrate that the conformations of LeuT-substrate complexes in lipid bicelles and in β-OG detergent micelles are nearly identical. Furthermore, using crystals grown in bicelles and the substrate leucine or the substrate analog selenomethionine, we find only a single substrate molecule in the primary binding site.

Figure 1

Experimental flow chart and lattice contacts in bicelle-based crystal forms. (a) Flow chart defining the detergents and ligands used during purification and crystallization. (b–e) Crystal packing for different crystals forms: C2 (LeuT-Leu; panel b) and P2 (LeuT-Leu; panel c), viewed perpendicular to the b axis; P2₁ (LeuT-Leu form A; panel d) and P2₁2₁2 (LeuT-SeMet; panel e), viewed perpendicular to the c axis and a axis, respectively.

Figure 2

LeuT crystal structures derived from DMPC-CHAPSO bicelles and n-octyl-β-D-glucopyranoside (β-OG) are similar. (a) Stereoview of α-carbon traces of the refined LeuT structures from the C2 bicelle crystal form (cyan) and the C2 β-OG crystal form (PDB code 2A65; magenta) following superposition of corresponding main chain atoms. The regions enclosed by solid, dashed and dotted lines define the TM6a (Pro241-Leu255), TM11-EL6 (Val465-Val483) and EL2 (Val154-Lys163) regions. (b) Electron density for TM6a in the C2 bicelle crystal form. Shown is electron density from a 2Fo-Fc map contoured at 1.5σ. Atoms in TM6a are in stick representation. (c) Close up of TM6a, the carboxyl terminus of TM11 and EL6 (d) and the carboxyl terminus of EL2 (e). Superpositions shown in panels c–e are derived from panel a.

Figure 3

Mapping detergent sites in LeuT. (a) The anomalous difference Fourier map for LeuT (blue) crystallized in n-heptyl-β-D-selenoglucoside (β-SeHG). The map is contoured at 5σ, depicted in green mesh and unambiguously shows the positions of bound detergent molecules. Seven β-SeHG molecules are modeled and shown in stick representation. The positions of two additional β-SeHG molecules are inferred based on strong anomalous difference peaks. Here we have only modeled the Se atoms (orange spheres) due to weak electron density for the headgroups and alkyl chains. Leu in the primary site is shown in stick representation (green) with several residues omitted for clarity. (b) The anomalous difference map for LeuT-β-SeHG viewed from extracellular side of the protein. (c) The chemical structure of β-SeHG. (d) The F_o-F_c difference electron density map for LeuT-Leu (P2₁ form A, chain A, orange) crystallized in bicelles. The protein was purified using the detergent n-dodecyl-β-D-selenomaltoside (C₁₂SeM). This map is displayed at 4.5σ, depicted in blue mesh. (e) The two peaks in panel d are in the similar positions as the alkyl chain of β-SeHG in LeuT-β-SeHG structure and the phosphocholine molecule (PC) in LeuT-Leu (C2 form) structure, respectively. Both β-SeHG and PC molecules are shown as stick representation. (f) The chemical structure of C₁₂SeM.

Figure 4

LeuT has a single high affinity substrate site in bicelles. (a) Electron density map for the primary substrate binding site and extracellular vestibule in the DMPC-CHAPSO bicelle C2 crystal form of the LeuT-Leu complex. The substrate leucine in the primary site (green) is in stick representation and two sodium ions are shown as black spheres. Key residues in the vestibule are in stick representation. The F_o-F_c difference electron density map is displayed at 3σ, depicted in blue mesh and calculated with the substrate leucine and the two sodium ions omitted (Leu and Na are shown for reference). (b) Close up of primary binding site and the vestibule. (c) Anomalous difference Fourier map for primary substrate site and extracellular vestibule derived from diffraction data measured from LeuT-SeMet (C2 form) crystals. The map is contoured at 5σ and 20σ and depicted in green and red mesh, respectively. SeMet was positioned in the primary site and shown in stick representation. Sodium ions Na1 and Na2 are illustrated as black spheres (d) Close up of primary binding site and the vestibule.