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   it is possible that these cores move with respect to each other upon
   ligand binding. The effect of any of these activation mechanisms in the
   protomer will then be modulated by the varying subunit interfaces in
   the different subtypes of the receptor, allowing intricate specificity
   in neuronal signal transmission.
   [187]Top of page

 §3§ Conclusions  §3§

   This crystal structure shows that molluscan AChBP is a homologue of the
   pentameric LGIC superfamily ligand-binding domains. It confirms the
   predicted Ig-topology, the location of the binding site at the subunit
   interface, the position of the MIR and the extensive data on the
   nicotinic ligand-binding residues.

   With this structure, we present the first detailed three-dimensional
   information about the fold and the arrangement of the nicotinic
   ligand-binding site. It also clarifies the arrangement of subunits in
   nicotinic muscle receptors by showing the relative positioning of the
   principal and complementary part of the ligand-binding site.

   The AChBP crystal structure provides an explanation for the role of the
   pentameric LGIC superfamily conserved residues. They stabilize the
   protomer structure by the formation of hydrophobic cores and packing of
   secondary structure elements. The crystal structure clarifies how the
   LGIC pentamers are built up, and how weakly the pentamer interfaces are
   conserved between LGICs, which has implications for the modes of
   action.

   It is unclear whether AChBP performs the allosteric and desensitization
   movements that are important for pentameric LGIC function. AChBP is a
   soluble protein with a natural role in the modulation of synaptic
   transmission and lacks a transmembrane channel^[188]27. Thus, ligand
   binding does not result in opening of a pore in AChBP, although some of
   the movements could be conserved in its evolution. Similarly, it is
   unclear whether non-competitive antagonist-binding sites are present in
   AChBP. Such questions can be addressed with chimaeric molecules and
   possibly by further crystallographic studies.

   This structure will be highly relevant for the numerous drug-design
   studies that are targeting the pentameric LGIC superfamily. The general
   structural knowledge on its folding will be applicable to the GABA,
   serotonin (5-HT[3]) and glycine receptor fields. It will help us to
   understand their ligand-binding characteristics and hence could have an
   impact on the development of, for example, anti-emetics aimed at the
   5-HT[3] receptor or the mood-defining drugs that target the GABA
   receptors. The availability of a three-dimensional description of the
   nicotinic ligand-binding site will be especially relevant for the
   design of new drugs against, for example, Alzheimer's disease, epilepsy
   and addiction to smoking.
   [189]Top of page

 §3§ Methods §3§

 §4§ Protein purification §4§

   AChBP was cloned into expression vector pPIC9 (without residue Leu 1)
   and overexpressed in yeast, Pichia pastoris GS115, according to the
   Invitrogen manual. After 4 days of induction the medium was collected,
   concentrated and dialysed against standard buffer (20 mM Tris–HCl
   buffer (pH 8.0), 150 mM NaCl and 0.02% (w/v) NaN[3]). The protein was
   purified by anion exchange (Poros50 HQ, MonoQ), and gel filtration
   (Superdex 200). It was dialysed against 50 mM HEPES buffer (pH 7.0)
   with 0.02% NaN[3] and concentrated to approx 20 mg ml^-1. N-terminal
   sequencing revealed that part of the pPIC9-encoded signal sequence is
   retained, before residue 2 (sequence EAEAYVEF). The experimental
   relative molecular mass was 26,544 (MALDI), approx 2K more than the
   calculated mass based on the sequence (24,649), owing to glycosylation
   at position Asn 66, as confirmed by deglycosylation experiments (data
   not shown).

 §4§ Crystallization §4§

   We grew the crystals at room temperature using the hanging-drop vapour
   diffusion technique. All drops contained 2  micro l of protein
   (10 mg ml^-1) and 2  micro l of reservoir solution (9–11% (w/v) PEG
   4000, 100 mM HEPES (pH 7.0), 50–200 mM CaCl[2] and 0.02% NaN[3]).
   Depending on the batch of protein and the CaCl[2] concentration, we
   obtained three crystal forms. Orthorhombic and monoclinic crystals
   appeared under high CaCl[2] concentration and were frequently twinned.
   The orthorhombic crystals (P2[1]2[1]2[1]) have the following cell
   constants: a = 120.6 Ã…, b = 137.0 Ã…, c = 161.5 Ã…, with two pentamer
   molecules per asymmetric unit (asu). The monoclinic crystals (P2[1])
   are very similar in morphology to the orthorhombic ones, but gave lower
   resolution data ( approx 3.3 Ã…), with the following cell constants: a =
   121.1 Å, b = 162.1 Å, c = 139.4 Å, beta = 90.13°, and four pentamers
   per asu. The tetragonal crystal form (P4[2]2[1]2) was obtained from a
   solution containing 11.5 (w/v) PEG 4000, 100 mM HEPES (pH 7.0), 150 mM
   CaCl[2] and 0.02% (w/v) NaN[3]. They have the following cell constants:
   a = b = 141.66 Ã…, c = 120.83 Ã… and one pentamer per asu. For MAD
   experiments, orthorhombic and monoclinic crystals were soaked in mother
   liquor solution containing 5 mM and 10 mM trimethyl-lead acetate (MePb)
   respectively for 5 days. Before flash-cooling, all crystals were
   gradually equilibrated against mother liquor with 30% glycerol.

   In all three space groups AChBP forms a decamer structure with 52
   symmetry, where 2 pentamers have contact with each other through a
   calcium-binding site, at the 'top' of the alpha 1 helix. This binding
   site (Asp 2 and Asp 5 from two protomers) is not conserved in the
   pentameric LGIC family. In the tetragonal space group the two-fold of
   the decamer coincides with a crystallographic two-fold. In solution,
   the AChBP protein behaves as a pentamer^[190]27.

 §4§ Structure determination and refinement §4§

   We collected native data (X11) and the Pb-1 data sets (BW7A) at the
   EMBL outstation at the DESY synchrotron in Hamburg and the Pb-2 data
   sets (BM14) at the ESRF, Grenoble ([191]Table 1). Data were processed
   with DENZO/SCALEPACK^[192]41 (native) or MOSFLM^[193]42/SCALA^[194]43
   software (Pb-1, Pb-2). The Pb sites, located at the interface of two
   pentamers, were found for both MAD sets by SOLVE^[195]44 and heavy atom
   parameters were optimized with SHARP^[196]45. NCS operators were found
   and refined with NCS6D and IMP^[197]46. Multi-crystal averaging was
   executed with DM-multi^[198]47 using amplitudes from the monoclinic,
   orthorhombic and native (tetragonal) data sets, and experimental phases
   from the orthorhombic and monoclinic MAD experiments. The model was
   built in O^[199]48 and refined with CNS^[200]49, against the tetragonal
   2.7 Ã… data. Refinement included partial fivefold NCS restraints, an
   overall anisotropic B factor and bulk solvent correction. The unusual
   vicinal disulphide bridge^[201]8, Cys 187–Cys 188, was visible in the
   electron density, but because of the limited resolution it was not
   analysed in detail. The final model contains 1,025 residues of AChBP
   pentamer, 5 HEPES molecules, 10 Ca^2+ ions and 15 water molecules. The
   entire AChBP pentamer is well ordered, except for the N-terminus seven
   residues (part of the signal sequence) and the last five C-terminal
   residues. In addition, the HEPES, loop region 155–160 and the sugar
   residues attached to residue Asn 66 are not well resolved in the
   electron density. Root-mean-square deviations from ideal geometry for
   bond distances and angles are 0.01 Å and 1.6°, respectively.

   [202]Top of page

 §3§ References §3§

   ------------------

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