Stability of ligand-binding domain dimer assembly controls kainate receptor desensitization
2009; Springer Nature; Volume: 28; Issue: 10 Linguagem: Inglês
10.1038/emboj.2009.86
ISSN1460-2075
AutoresCharu Chaudhry, Matthew C. Weston, Peter Schuck, Christian Rosenmund, Mark L. Mayer,
Tópico(s)Cellular transport and secretion
ResumoArticle2 April 2009free access Stability of ligand-binding domain dimer assembly controls kainate receptor desensitization Charu Chaudhry Charu Chaudhry Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD, USA Search for more papers by this author Matthew C Weston Matthew C Weston Departments of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Peter Schuck Peter Schuck Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science, NIBIB, NIH, DHHS, Bethesda, MD, USA Search for more papers by this author Christian Rosenmund Christian Rosenmund Departments of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Mark L Mayer Corresponding Author Mark L Mayer Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD, USA Search for more papers by this author Charu Chaudhry Charu Chaudhry Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD, USA Search for more papers by this author Matthew C Weston Matthew C Weston Departments of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Peter Schuck Peter Schuck Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science, NIBIB, NIH, DHHS, Bethesda, MD, USA Search for more papers by this author Christian Rosenmund Christian Rosenmund Departments of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Mark L Mayer Corresponding Author Mark L Mayer Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD, USA Search for more papers by this author Author Information Charu Chaudhry1,‡, Matthew C Weston2,‡, Peter Schuck3, Christian Rosenmund2 and Mark L Mayer 1 1Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, NICHD, NIH, DHHS, Bethesda, MD, USA 2Departments of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 3Dynamics of Macromolecular Assembly, Laboratory of Bioengineering and Physical Science, NIBIB, NIH, DHHS, Bethesda, MD, USA ‡These authors contributed equally to this work *Corresponding author. Porter Neuroscience Research Center, NIH, Bldg 35 Room 3B 1002, 35 Lincoln Drive, Bethesda, MD 20892 3712, USA. Tel.: +301 496 9346 (lab 9347); Fax: +301 496 2396; E-mail: [email protected] The EMBO Journal (2009)28:1518-1530https://doi.org/10.1038/emboj.2009.86 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info AMPA and kainate receptors mediate fast synaptic transmission. AMPA receptor ligand-binding domains form dimers, which are key functional units controlling ion-channel activation and desensitization. Dimer stability is inversely related to the rate and extent of desensitization. Kainate and AMPA receptors share common structural elements, but functional measurements suggest that subunit assembly and gating differs between these subtypes. To investigate this, we constructed a library of GluR6 kainate receptor mutants and directly measured changes in kainate receptor dimer stability by analytical ultracentrifugation, which, combined with electrophysiological experiments, revealed an inverse correlation between dimer stability and the rate of desensitization. We solved crystal structures for a series of five GluR6 mutants, to understand the molecular mechanisms for dimer stabilization. We demonstrate that the desensitized state of kainate receptors acts as a deep energy well offsetting the stabilizing effects of dimer interface mutants, and that the deactivation of kainate receptor responses is dominated by entry into desensitized states. Our results show how neurotransmitter receptors with similar structures and gating mechanisms can exhibit strikingly different functional properties. Introduction Ionotropic glutamate receptors (iGluRs) mediate excitatory synaptic transmission by coupling the free energy of agonist binding to the opening and desensitization of a transmembrane ion channel (Gouaux, 2004; Mayer, 2006; Hansen et al, 2007). Central to the function of the 18 iGluR genes that form the AMPA, kainate and NMDA receptor families of ligand-gated ion channels is a structural unit formed by a dimer assembly of the ligand-binding domains (Armstrong and Gouaux, 2000; Mayer et al, 2001; Furukawa et al, 2005; Nanao et al, 2005). This dimer undergoes conformational changes driven by agonist-binding energy, producing transitions between the resting, conducting, and desensitized states of the ion channel. The rates of transitions between these states, which are finely tuned at individual excitatory synapses to allow information processing in the brain over a wide spectral bandwidth (Geiger et al, 1995), are controlled by intermolecular contacts in the dimer assembly (Sun et al, 2002; Horning and Mayer, 2004). An understanding of this process, which is much less well understood in kainate and NMDA receptors, is a key requirement for resolving the role of individual iGluR subtypes in synaptic plasticity. Following release of neurotransmitter by the presynaptic cell, receptors in the postsynaptic membrane bind glutamate in a two-domain clamshell-shaped structure (LBD) formed by S1 and S2 polypeptide segments (Stern-Bach et al, 1994; Kuusinen et al, 1995). Structural studies on AMPA receptor GluR2 S1S2 have shown that activation occurs on glutamate binding through rotation of domain 2 and closure of the clamshell. A critical inter-subunit interface, formed between individual protomers in a dimer pair through the rear surface of domain 1, allows domain closure to place a torque on the membrane-embedded ion channel, causing it to open (Armstrong and Gouaux, 2000). A major advance in our understanding of AMPA receptor gating was the serendipitous discovery, by construction of chimeric receptors, that the GluR3 L485Y mutation blocks desensitization (Stern-Bach et al, 1998). Structural analysis for the equivalent GluR2 L483Y mutant revealed that the tyrosine side chain stabilizes dimer assembly by forming a cation–π interaction with a lysine side chain in the dimer partner subunit (Sun et al, 2002). This finding has profoundly influenced our understanding of AMPA receptor gating, and led to a model in which the domain 1 interface is under strain in the glutamate-bound state. During desensitization the interface ruptures, allowing the channel to close even with the agonist bound (Armstrong et al, 2006). Paradoxically, the genes for GluR5–GluR7 subtype kainate receptors encode an aromatic amino acid at the position equivalent to GluR2 L483Y, but desensitize rapidly and nearly completely in response to glutamate (Schiffer et al, 1997; Swanson et al, 1997). Functional analysis of the LBD dimer interface and recent crystal structures of kainate receptor dimer assemblies, which bear striking structural similarity to their AMPA receptor counterparts, have not resolved this puzzle. It is to be noted that numerous differences exist between the two receptor families in their sensitivities to allosteric ligands such as cyclothiazide and concanavalin A (Partin et al, 1993; Wong and Mayer, 1993; Yamada and Tang, 1993), and to external ions that modulate desensitization (Bowie, 2002; Bowie and Lange, 2002; Plested et al, 2008). An approach taken by several groups has been to rebuild the dimer interface of kainate receptors by introducing AMPA receptor residues, in an attempt to recreate the non-desensitizing GluR2 L483Y phenotype (Fleck et al, 2003; Zhang et al, 2006; Weston et al, 2006b). However, with the exception of disulphide cross-links, which perturb the dimer interface, render glutamate a partial agonist, and disrupt trafficking to the plasma membrane, all attempts to generate non-desensitizing kainate receptor mutants through rational design have ultimately fallen short, having greater effects on the rate rather than the extent of desensitization (Swanson et al, 1997; Stern-Bach et al, 1998; Fleck et al, 2003; Yelshansky et al, 2004; Priel et al, 2006; Zhang et al, 2006; Weston et al, 2006b). Here we explore the reason for this and test two plausible mechanisms that have important consequences for understanding kainate receptor function. The first is that the kainate receptor desensitized state acts as a deep energy well, competing with strengthening of the dimer assembly obtained by introduction of AMPA receptor residues. To address this, we measured rates of onset and recovery from desensitization using electrophysiological assays for a family of dimer interface mutants. An alternative mechanism would be that the dimer interface of kainate receptors is intrinsically less stable than that of AMPA receptors. These two possibilities represent extremes, which are not mutually exclusive. As the driving force for desensitization arises from strain imposed on LBD dimers by the ion channel, this complicates comparisons between AMPA and kainate receptor mutants based solely on electrophysiological studies. To avoid this, we directly measure the Kd for dimer formation by isolated LBDs for GluR6 in the absence of the ion channel using analytical ultracentrifugation (AUC). To test whether mutants in the dimer interface sense different local environments in the two receptors, we solved a library of five high-resolution crystal structures for GluR6 mutants, together with the structure for the wild-type GluR6 dimer crystallized under identical conditions. Our results indicate that even following extensive engineering, the stability of kainate receptor dimers is at most one half of that of their AMPA counterparts, and that even if it were possible to generate dimers as stable as those for GluR2 L483Y, these would be insufficient to block kainate receptor desensitization because of the deep energy well of the desensitized state. We propose that this intrinsic difference in dimer stability contributed to the evolution of subtype-specific allosteric regulators, for example, the recently described Na+- and Cl−-binding sites in kainate receptors (Plested and Mayer, 2007; Plested et al, 2008). Results Desensitization is regulated by clusters of dimer interface residues Analysis of dimer crystal structures in the protein data bank for GluR2, GluR5, and GluR6 reveals that, although they share a similar dimer structure, conserved clusters of residues that contribute to the dimer interface differ in the two receptor families (Figure 1). Structure-based sequence alignments reveal that in addition to earlier identified amino-acid differences in α-helices D, F, and J, which flank the GluR2 L483Y tyrosine mutant that blocks AMPA receptor desensitization (Figure 1A), helix B harbors conserved Lys and His side chains in AMPA receptors, which are absent in kainate receptors. Mutation of these residues to their AMPA counterparts would be expected to alter the electrostatic environment in the dimer interface, and, together with earlier reported mutants of residues in helices D, F, and J, facilitate intermolecular interactions with the native (GluR6 Y490) tyrosine side chain in kainate receptors (Zhang et al, 2006; Weston et al, 2006b). To examine the influence of these residues on dimer stability, we created a library of 10 GluR6 mutants, which, using different combinations, progressively switch the sequence of seven amino acids in helices B, D, F, and J to the sequence found in GluR2. The mutants were T441K, I442H, K494E, K665R, I749L, Q753K, and E757Q; for brevity we use dashes to indicate the wild-type GluR6 sequence for mutants in which a limited number of amino acids was changed; for example, the GluR6 K665R mutant is designated - - -R- - -. These mutants were then assayed for changes in gating using full-length GluR6 (Figure 2), and for changes in dimer stability using analytical ultracentrifugation for the isolated LBDs (Figure 3). Figure 1.Conserved clusters of residues differ in the dimer interface of AMPA and kainate receptors. (A) Amino-acid sequence alignment for AMPA and kainate receptor gene families; dimer interface residues exchanged between GluR2 and GluR6 are indicated by Δ; additional residues that play key roles in the effects of individual mutations are indicated by *; cylinders above the alignment indicate location of α-helices B, D, F, and J in GluR6 crystal structures; + indicates the L/Y switch in helix D. (B) Ribbon diagram for wild-type GluR6 shows the location of the critical Tyr490 side chain, surrounded by residues exchanged between GluR2 and GluR6, drawn as gold- and cyan-colored CPK spheres for the pair of subunits in a dimer assembly (a stereo view is shown in Supplementary Figure 1). Download figure Download PowerPoint Figure 2.Kinetic analysis for a library of GluR6 dimer interface mutants. (A) Responses of outside-out patches to 100-ms applications of 10 mM glutamate are shown for wild type and three mutants. The rate of desensitization (kdes) is fastest for wild type, ∼200-fold slower for -HERLK-, whereas - - -R- - - and -HE-LK- produce intermediate kinetics. (B) Responses to 7-s applications of glutamate show the extent of desensitization for -HE-LK- and -HERLK-, highlighting the impact of the K665R mutation in the -HE-LK- background. (C) The extent of desensitization measured at 100 ms (black) and 7 s (red). (D) Rate of onset of desensitization (black bar) and recovery (grey bar), for wild-type GluR6 and the library of 10 dimer interface mutants; error bars indicate mean±s.e.m. Download figure Download PowerPoint Figure 3.Dimer formation for GluR6 LBDs measured by analytical ultracentrifugation. (A) c(s) distributions from sedimentation velocity (SV) runs for -HERLK- (1.7 mg ml−1), -HE-LK- (1.9 mg ml−1) and - - -R- - - (2 mg ml−1); concentrations reported in parentheses are derived from peak integration (see also Supplementary Figure 2). Peak positions reflect sedimentation of rapidly interconverting monomer–dimer systems. The ∼3.6 S peak for -HERLK- reflects stronger association of this mutant compared with the 2.8 and 2.9 S peaks for -HE-LK- and - - -R- - -, respectively. (B) Dependence of the weight-average sedimentation coefficient sw on loading concentration for -HERLK-, -HE-LK- and - - -R- - - (symbols), and best fits with a binding isotherm for a monomer–dimer equilibrium (solid lines) with Kd values of 41.2 μM (1σ confidence interval 37–45 μM), 416 μM (1σ 370–460), 321 μM (1σ 307–333) for -HERLK-, -HE-LK- and - - -R- - -, respectively. (C) Representative sedimentation equilibrium profile for -HERLK- derived from a global analysis of data at a range of loading concentrations and rotor speeds, using a monomer–dimer model with a Kd of 102 μM (1σ 85–135). The black line indicates the model used to fit the data, red dots indicate experimental measurements, and dashed lines represent the best-fit populations of monomer and dimer; residuals of the fit are shown below the graph. (D) The free energy change for dimerization ΔGdimer plotted against the free energy change for onset of desensitization relative to wild type (−RT ln kdes/kwt) for each mutant (symbol and error bars). The dotted line shows the best fit from total least-squares optimization, suggesting a linear relationship in which stabilization of the dimer assembly slows the rate of onset of desensitization. A full colour version of this figure is available at The EMBO Journal online. Download figure Download PowerPoint Successively introducing combinations of AMPA receptor residues into the wild-type GluR6 produced progressively slower rates of onset of desensitization (kdes) with rank-order responses to 10 mM glutamate of the wild-type GluR6 284±27 s−1, > - - - -LK- 48.9±8.5 s−1, > - - -R- - - 36.2±9.4 s−1, >- -E-LK- 17.8±2.6 s−1, >-HE-LK- 12.8±1.2 s−1, > - -ERLK- 3.2±0.4 s−1, >-HERLK- 1.5±0.1 s−1 ≈ KHERLK- 1.8±0.1 s−1. The most effective combination of mutations, -HERLK-, slowed kdes 190-fold compared with wild-type GluR6, producing a strong attenuation of desensitization measured 100 ms after the start of glutamate application, from 99.4±0.2% for wild-type GluR6 to 20±2% for -HERLK- (Figure 2A); however, when glutamate application was increased to 7 s, the extent of desensitization for -HERLK- at equilibrium increased to 81±3% (Figure 2B). This is in striking contrast to the nearly complete block of desensitization produced by GluR2 L483Y. Our analysis suggests that in -HERLK- the mutations K665R near the base of helix F and the pair I749L/Q753K in helix J act independently of each other, as reversion of each site to the wild-type sequence led to a similar 8- to 10-fold increase in the extent (Figure 2C) and rate of desensitization (Figure 2D). The mutants tested had only small effects on the rate of recovery from desensitization (krec), measured using twin pulse applications of glutamate (Figure 2D, Table I), and krec varied 6000 ND 8000 ND <2.8 — 284±27 (7) 506±43 (6) 99.2±0.4 (7) 0.46±0.08 (6) Kd-SE and Kd-SV are dimer dissociation constants determined from sedimentation equilibrium and velocity experiments, respectively; error estimates are reported as 1σ confidence intervals in parentheses and calculated as described in Materials and methods; ND signifies not determined. Kd's for - -E-LKQ and wild-type GluR6 are lower limits determined as described previously. ΔGdd SE and SV are the corresponding ΔG of dimer dissociation, calculated as ΔG=−RT ln Kd (R=1.987 cal(mol K)−1); T=277 or 298 K for SE and SV experiments, respectively). kdes, kdeact, % des, and krec are the rates of desensitization, deactivation, extent of desensitization, and rate of recovery from desensitization as measured by fast-solution exchanges; measurements are the mean±s.e.m., with the number of observations in parentheses. Kd's for - -E-LKQ and wild-type GluR6 are lower limits determined as described previously. KHERLK and Q mutations correspond to the following changes in full-length GluR6, respectively: T441K, I442H, K494E, K665R, I749L, Q753K, and E757Q. Dimer mutants promote co-assembly of GluR6 ligand-binding domains Sedimentation velocity (SV) and sedimentation equilibrium (SE) analytical ultracentrifugation experiments were performed for the isolated LBDs of a library of eight GluR6 mutants, designed on the basis of electrophysiological results. As reported earlier, for both wild-type GluR6 and - -E-LKQ, dimer formation is too weak to be determined by either approach (Weston et al, 2006b), and error analysis indicates a lower limit of 6–8 mM for the dimer Kd. In contrast, the dimerization of each of the eight mutants was sufficiently strong to be detected by both SV and SE, yielding Kd values with very similar rank order as predicted from electrophysiological analysis (Table I). Examples of the SV approach, showing the c(s) sedimentation coefficient distribution at 20°C for -HERLK-, -HE-LK-, and - - -R- - -, at similar concentrations (Figure 3A), exhibit the expected behaviour for a monomer–dimer assembly in equilibrium (Schuck, 2000), with rapid interconversion on the sedimentation time-scale (Supplementary Figure 2). It is evident from visual inspection of overlaid c(s) distributions that -HERLK stabilizes dimer formation to a greater extent, judged by an increase in the sedimentation coefficient, relative to - - -R- - - and -HE-LK-, which behave similarly. The isotherm of weight-average sedimentation coefficients (sw) as a function of protein concentration followed the mass action law for a monomer–dimer equilibrium, yielding best-fit Kd values of 41.2, 321, and 416 μM for -HERLK-, - - -R- - -, and -HE-LK-, respectively (Figure 3B). In parallel, SE experiments at 4°C were conducted and global non-linear least squares fits of these datasets to a monomer–dimer model allowed independent determination of the dimerization Kd. The results from both analyses were in good agreement (Table I), with Kd values for SV ∼2-fold lower than those measured by SE (see Supplementary data). When the free energy of dimer dissociation is plotted against the rate constant for onset of desensitization measured by electrophysiological analysis (Figure 3D), an almost linear relationship emerges, indicating that dimer stability is a major determinant of desensitization for kainate receptors. Dimer mutant crystal structures To define the molecular mechanisms underlying changes in dimer stability and attenuation of desensitization by these mutants, a structural library of GluR6 dimers in their active conformation was determined for the glutamate complexes of -HERLK-, -HE-LK-, - - -RLK-, - - -R- - -, and wild type crystallized in the presence of a physiological concentration of NaCl, with data for Bragg spacings ranging from 1.5 to 1.32 Å (Table II). A key finding in all of the structures was that Na+ and Cl− were present in the allosteric ion-binding sites, as found earlier for GluR5 dimer crystal structures with the partial agonist kainate (Plested and Mayer, 2007; Plested et al, 2008). As these ions do not bind to AMPA receptors, this indicates that, despite the introduction of up to six GluR2 residues into GluR6, the dimer interface maintains unique properties characteristic of kainate receptors. The dimer interface buries a similar solvent accessible surface of 1164, 1160, 1151, 1121, and 1204 A2 per subunit for -HERLK-, -HE-LK-, - - -RLK-, - - -R- - -, and wild-type GluR6, respectively, and superposition of the dimer assemblies using domain 1 (D1) Cα coordinates gave r.m.s. deviations of <0.2 Å, indicating that despite profound differences in their electrophysiological and biophysical properties, these mutants have nearly identical structures to wild-type GluR6. However, because the crystals diffract to high resolution, we were able to detect local conformational differences in each structure that explain the mutational effects on dimer stability (Figure 4). The tyrosine side chains on the exposed face of helix D move upwards by 1.2 and 1.3 Å in the pair of subunits in -HERLK- compared with wild-type GluR6; this movement occurs without a change in the intermolecular distance between helices D and J measured using the Cα coordinates of Tyr490 and Ile749, and instead results from both a rotation of the aromatic ring and a change in the χ1 side-chain dihedral angle. This movement has a profound effect on cation–π interactions made by Tyr490, which is a key determinant of the high dimer stability in the GluR2 L483Y mutant (Sun et al, 2002). To analyze the strength of cation–π interactions (Gallivan and Dougherty, 1999), we used the program CAPTURE. In wild-type GluR6, there are intra-subunit cation–π interactions between Lys494 and Tyr490 in the same subunit (−1.31 and −2.34 kcal mol−1 for subunits A and B, respectively), which are replaced by new intermolecular cation–π interactions with the mutant Lys753 side chain in -HERLK- (−2.04 kcal mol−1) and -HE-LK- (−2.19 kcal mol−1). Figure 4.High-resolution crystal structures for GluR6 LBD mutants. (A) Side chains drawn in stick representation are shown for wild-type GluR6, - - -R- - -, - - -RLK-, -HE-LK- and -HERLK- following least-squares superposition of dimer assemblies using D1 Cα coordinates; a stereo view with CPK spheres for -HERLK- is shown in Supplementary Figure 1. (B) σA-weighted 2mFo−DFc electron density maps contoured at 1σ for amino-acid side chains surrounding the native Tyr490 residue; for visualization side chains have been rotated from their orientation in the dimers. (C) Interactions between Tyr490 on one subunit with I749L and Q753K on helix J of the dimer partner for wild-type GluR6 (grey) and -HERLK- (gold). The van der Waals radii of Ile749 for wild-type GluR6 show a steric clash (top panel) with those for Tyr490 in the -HERLK- structure, which is eradicated on mutation to a Leu (bottom panel). (D) Interdomain interactions between helices B and J on one subunit and helices D and F on the other for HERLK and wild-type GluR6. Van der Waals interactions (distance <4 Å) are represented by a solid line connecting partner amino acids; hydrogen bonds by arrows pointing in the direction of the hydrogen bond acceptor; electrostatic (cation–π and salt-bridge) interactions by a thick line; water-mediated interactions with a dashed line; grey and gold indicate bonds for wild-type GluR6 and -HERLK-, respectively. Download figure Download PowerPoint Table 2. Data collection and refinement statistics Data set WTR6 - - -R- - - - - -RLK- -HE-LK- -HERLK- -HERLKQ Data collection Space group P21 P21 P21 P21 P21 P21 Unit cell a, b, c (Å) 51.2, 114.2, 52.4 51.0, 113.5, 52.0 51.1, 113.5, 52.2 51.2, 114.2, 52.4 50.9, 113.7, 51.9 51.1, 113.8, 52.1 a=γ, β 90, 115.3 90, 115.2 90, 115.1 90, 115.3 90, 115.3 90, 115.3 Number per a.u. 2 2 2 2 2 2 Wavelength (Å) 1.0000 1.0000 1.0000 1.0000 1.0000 0.97930 Resolution (Å)a 30–1.38 (1.43) 30–1.3 (1.35) 30–1.5 (1.55) 30–1.37 (1.42) 35–1.32 (1.37) 40–1.2 (1.24) Unique observations 102 842 129 531 83 975 109 022 124 266 149 021 Mean redundancyb 4.9 (4.8) 6.3 (6.0) 2.8 (2.4) 7.3 (7.2) 2.8 (2.6) 4.6 (2.8) Completeness (%)b 94.2 (84.0) 99.1 (91.4) 97.9 (94.0) 94.3 (89.3) 94.7 (88.6) 98.6 (86.5) Rmergeb,c, b,c 0.043 (0.315) 0.039 (0.309) 0.053 (0.347) 0.038 (0.241) 0.044 (0.369) 0.064 (0.239) I/σ(I)b 15.0 (2.4) 30.6 (3.4) 17.6 (2.8) 18.0 (2.9) 24.0 (4.1) 22.5 (3.4) Refinement Resolution (Å) 28.71–1.38 29.26–1.30 29.53–1.50 28.69–1.37 34.5–1.32 35.9–1.24 Protein atoms (AC)d 4276 (223) 4302 (227) 4275 (271) 4364 (275) 4331 (246) 4680 (560) Glutamate atoms 20 20 20 20 20 20 Ions/Cl/Na atoms 1/4 3/2 2/3 1/3 2/4 5/2 Water atoms 581 665 721 694 618 724 Rwork/Rfree (%)e 15.21/17.49 14.63/16.76 14.74/17.64 14.93/17.60 15.61/17.89 14.59/16.50 r.m.s. deviations Bond lengths (Å) 0.012 0.016 0.010 0.010 0.010 0.017 Bond angles° 1.18 1.39 1.13 1.14 1.11 1.39 Mean B-values (Å2) Protein overall 20.37 14.94 15.58 18.37 17.37 15.62 MC/SCf 17.08/23.5 12.46/17.30 12.85/18.17 15.45/21.11 14.89/19.74 13.01/17.88 Glutamate 12.65 7.13 7.45 9.50 8.91 7.61 Cl/Na 15.2/25.3 16.8/15.1 11.9/19.2 12.9/23.7 16.7/29.3 18.35/16.6 Water 32.3 26.6 28.4 31.2 31.2 27.6 Ramachandran %g 98.0/0 98.2/0 98.19/0 98.22/0 98.0/0 98.2/0 a Values in parentheses indicate the low-resolution limit for the highest-resolution shell of data. b Values in parentheses indicate statistics for the highest-resolution shell of data. c Rmerge==(Σ∣II–〈II〉∣)/ΣI∣II∣, where 〈II〉 is the mean II over symmetry-equivalent reflections. d Alternate conformations. e Rwork=(Σ∣∣Fo∣–∣Fc∣∣)/Σ∣Fo∣, where Fo and Fc denote observed and calculated structure factors, respectively; 5% of the reflections were set aside for the calculation of the Rfree value. f Main chain/side chain. g Preferred/disallowed conformations. Electron density maps were of high quality for Tyr490 in all structures, whereas for Q753K, side-chain electron density, assessed by real space correlation coefficients and by visual inspection of omit maps, was best modelled by alternative conformations in several constructs (Figure 4B and Supplementary Figure 3). In -HERLK-, the replacement of Ile442 by the large positively charged imidazole ring of histidine biases the conformation of Lys753, increasing the probability for making a cation–π interaction with Tyr490. Indeed, in the library of constructs with the Q753K mutation, the side chain was
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