A network of conserved intramolecular contacts defines the off-state of the transmembrane switch mechanism in a seven-transmembrane receptor.

Activation of the rhodopsin-like 7-transmembrane (7-TM) receptors requires switching interhelical constraints that stabilize the inactive state to a new set of contacts in the activated state, which binds the cognate G-protein. The free energy to drive this is provided by agonist binding, which has higher affinity to the active than to the inactive conformation. We have sought specific interhelical constraint contacts, using the M(1) muscarinic acetylcholine receptor as a model. Histidine substitutions of particular groups of amino acids, in transmembrane domains 3, 6, and 7, created high-affinity Zn(2+) binding sites, demonstrating the close proximity of their side chains in the inactive state. Alanine point substitutions have shown the effect of weakening the individual intramolecular contacts. In each case, the acetylcholine affinity was increased, implying promotion of the activated state. These amino acids are highly conserved throughout the 7-TM receptor superfamily. We propose that they form an important part of a network of conserved interhelical contacts that defines the off-state of a general transmembrane switch mechanism.

Mutations which cause agonist-independent activation of 7-TM 1 receptors have suggested the presence of structural constraints; for instance, a salt bridge between TM 3 and TM 7 may stabilize the ground-state conformation of rhodopsin (1) and the ␣ 1B receptor (2). Recently, we identified a patch of amino acids in TM 3 of the M 1 mAChR, including the highly conserved residues Leu 116 and Ser 120 , whose mutation increased ACh affinity and caused constitutive activation (3,4). We proposed that this follows the deletion of specific intramolecular contacts.
Although the high-resolution structures of the 7-TM receptors are unknown, a general model of the ␣-carbon backbone has been published, based on sequence analysis in the context of a low-resolution electron crystallographic structure of rhodopsin (5,6). Interactions between highly conserved amino acids within the receptor core were proposed to mediate receptor conformational changes. In reference to the model, potential contacts for Leu 116 are Phe 374 (TM 6), Asn 414 (TM 7), and Tyr 418 (TM 7), while Ser 120 may contact Tyr 208 (TM 5). Muta-tions of Phe 374 cause constitutive activation of the M 5 mAChR (7), whereas Asn 414 and Tyr 418 have proved important for signal transduction in all of the 7-TM receptors in which they have been investigated (8 -14). The locations of these residues are indicated in Fig. 1, a and b. We have used polymerase chain reaction mutagenesis to make combinatorial histidine substitutions of these residues, creating Zn 2ϩ binding sites which have allowed us to explore this network of contacts in the M 1 mAChR.

EXPERIMENTAL PROCEDURES
Mutagenesis and Binding Studies-Mutants were constructed by a polymerase chain reaction method and validated by di-deoxy sequencing. Mutant receptors were subcloned into the pCD expression vector and expressed transiently in COS-7 cells by electroporation as described (4,(15)(16)(17). Binding studies were conducted in 20 mM Na-Hepes buffer plus 100 mM NaCl, pH 7.5, for 2 h at 30°C. (Ϫ)-N-[ 3 H]methylscopolamine ([ 3 H]NMS) binding curves were fitted to a one-site model to yield a total concentration of binding sites, R t (expressed relative to a wild-type control in each transfection) and an affinity constant, K NMS . Expression of the wild-type M 1 mAChR varied from 0.7 to 1.2 pmol/mg of protein in different transfections. The affinity of [ 3 H]NMS for the wild-type receptor was 1.0 Ϯ 0.05 ϫ 10 10 M Ϫ1 . Inhibition curves for ACh and ZnCl 2 were fitted to the Hill equation, and the binding constants, K ACh and K Zn , were corrected for the Cheng-Prusoff shift, as necessary (4,15). K ACh for the wild-type M 1 mAChR was 1.1 Ϯ 0.1 ϫ 10 5 M Ϫ1 .
Functional Studies-Phosphoinositide (PI) dose-response curves to ACh were determined as described (4,15) and fitted to a four-parameter logistic function, yielding an EC 50 value and a maximum response (E max ). E max values are expressed relative to wild-type control in each transfection.

Ligand Binding and Receptor
Expression-With one exception (N414H-Y418H), the single, double, and triple His mutants were expressed at between 30 and 170% of the wild-type level in COS-7 cells, assayed by the specific binding of a highaffinity antagonist [ 3 H]NMS (Table I). Most of the mutations caused less than a 3-fold change in [ 3 H]NMS affinity. However, the F374H mutation caused a 7-fold reduction, which was partly reversed by the addition of a second His at position 116 or 414. The N414H-Y418H mutant gave 1% of wild-type expression, whereas its affinity was reduced by more than 10fold. Both of these effects were substantially reversed by the incorporation of a third His at position 116 or 374.
The mutation of F374H reduced ACh affinity by 17-fold (Table I), whereas mutation of Asn 414 and Tyr 418 to His (Table  I) increased it by between 4-and 15-fold. The ACh affinities of the double and triple mutants were similar to the geometric means of the constituent single mutants.
The binding and functional data (Table I) suggested that, with the exception of N414H-Y418H, the His mutations caused only minor perturbations of the ground-state structure of the M 1 mAChR. Thus, probing the substituted structures with Zn 2ϩ ions, and determination of the chelate effect, should give information about the spatial proximities of the parent residues.
Zn 2ϩ Inhibition of Antagonist Binding-Zn 2ϩ binding was assayed by inhibition of the binding of [ 3 H]NMS (2 ϫ K d concentration). The apparent K d of Zn 2ϩ for the wild-type receptor was 0.72 mM. The results are exemplified in Fig. 2a and sum-marized in Fig. 3a, which are expressed relative to the wildtype receptor.
Single histidine substitutions of Ser 120 , Tyr 208 , Phe 374 , Asn 414 , and Tyr 418 gave less than 6-fold increases in the Zn 2ϩ affinity. However, the mutation of Leu 116 increased the affinity by 14-fold.
The double histidine mutant L116H-S120H, with a favorable i and i ϩ 4 helical spacing, displayed a 43-fold increase in Zn 2ϩ affinity (Fig. 2a). The contribution to the free energy arising from cooperativity of the two histidines, calculated from the chelate effect (⌬⌬G o chelate ), was 2.6 kJ/mol (legend to Fig. 3). L116H-F374H and L116H-N414H yielded 71-and 32-fold increases in affinity, respectively, also giving positive chelate effects. However, L116H-Y208H, L116H-Y418H, as well as the combinations of S120H with Y208H, F374H, N414H, and Y418H did not show increased Zn 2ϩ affinity relative to the single mutants and gave no chelate effect. The double mutations F374H-N414H and F374H-Y418H increased the affinity of Zn 2ϩ by 8 -14-fold (Fig. 3a).
The triple histidine mutant generated by the addition of a third His at position 374 to L116H-S120H gave a further in- crease in Zn 2ϩ affinity of 5.7-fold, to 245-fold the wild-type value (⌬⌬G o chelate ϭ 6.3 kJ/mol) (Fig. 2a). In contrast, adding a third His at positions 208, 414, or 418 had little or no further effect. Combining L116H-F374H with N414H gave a 7-fold additional increase, to 504-fold the wild-type value (⌬⌬G o chelate ϭ 7.9 kJ/mol). Although it was impossible to measure the Zn 2ϩ affinity of the poorly expressed double mutant N414H-Y418H, the triple mutant F374H-N414H-Y418H showed a 75-fold increase relative to the wild-type receptor. This was 6-and 16fold greater than the F374H-N414H or F374H-Y418H mutants, corresponding to a ⌬⌬G o chelate of 5.0 kJ/mol (Fig. 3, a and  b). An interaction between these residues is also supported by the rescue of receptor expression and affinity in the triple mutant relative to the N414H-Y418H mutant (Table I).
Zn 2ϩ inhibition of binding of the tertiary antagonist [ 3 H]quinuclidinyl benzilate was also tested for selected mutants, giving results similar to those measured by [ 3 H]NMS. 2 Increasing the concentration of [ 3 H]NMS from 2 K d to 6 K d or 18 K d shifted the inhibition curves to higher concentrations of Zn 2ϩ (Fig. 2b) but did not reduce the maximum inhibition, thus showing high negative cooperativity of inhibition. None of these mutants increased the affinity of Ni 2ϩ more than 8-fold. 2 The inhibition of [ 3 H]NMS binding to the histidine mutants by Zn 2ϩ at the IC 50 concentration was reversed by subsequent addition of 1 mM excess of EDTA to chelate the Zn 2ϩ ions. This had less effect on the nonspecific binding of Zn 2ϩ to the wildtype receptor (Fig. 2c). These results indicated that relatively specific Zn 2ϩ binding sites were created and that the Zn 2ϩ binding to the specific sites was reversible.
Zn 2ϩ Effects on the PI Response-Zn 2ϩ (100 -300 M) inhibited the PI response evoked by ACh at the L116H-F374H mutant (70% inhibition of the effect of 10 Ϫ4 M ACh at 300 M Zn 2ϩ ) while having little effect on the wild-type receptor (20% inhibition of the effect of 10 Ϫ5 M ACh at 300 M Zn 2ϩ ) or the F374H and L116H mutants (less than 20% inhibition of the effect of 10 Ϫ4 M ACh at 300 M Zn 2ϩ ). However, Zn 2ϩ (10 -100 M) also doubled the basal PI signal in untransfected COS-7 cells, indistinguishably from cells transfected with the inactive L116H-N414H, F374H-N414H, and L116H-F374H-N414H mutants. The occurrence of this background of nonspecific stimulation made it impossible to quantitate the effects of Zn 2ϩ on the ACh-induced PI response, and these experiments were not pursued further. DISCUSSION Three of the triple His mutants showed strong positive cooperativity of Zn 2ϩ binding by histidine (Fig. 3b), corresponding to a ⌬⌬G o chelate in the range of 5.0 -7.9 kJ/mol. The K d values for Zn 2ϩ of these triads (L116H-S120H-F374H, 2.9 M; L116H-F374H-N414H, 1.4 M; F374H-N414H-Y418H, 9.5 M) are comparable with those reported for triple-His mutants in the NK-1 (18) and -opioid receptors (19) and suggest the successful creation of high-affinity Zn 2ϩ binding sites. The Hill coefficients of Zn 2ϩ inhibition of [ 3 H]NMS binding to these triple mutants were close to 1.0, consistent with the ligation of a single Zn 2ϩ ion. The inhibition mechanism appeared near competitive, over the concentration ranges studied. It is likely that the introduction of a positively charged Zn 2ϩ ion into the central cleft of the receptor strongly disfavors the binding of the positively charged radiolabeled antagonist, even though the altered residues do not overlap with the primary ligand binding residues (Fig. 1a).
The ␣-carbons of the residues composing the high-affinity triads must be separated by less than 13 Å to allow the corresponding imidazole side chains to coordinate a metal ion (20).  mAChR, a network of interactions exists between amino acid side chains, centered on Leu 116 (TM 3)-Phe 374 (TM 6)-Asn 414 (TM 7) and supported by Ser 120 (TM 3) and Tyr 418 (TM 7), which turn away from Leu 116 and Asn 414 by 40 degrees of arc (Fig. 4). In contrast, it seems unlikely that Tyr 208 (TM 5) is close enough to Ser 120 (TM 3) to form a hydrogen bond; in the TSH receptor, the homologous tyrosine has been proposed to make a hydrogen bond with a carbonyl oxygen in the peptide backbone of TM6 (21). Ala substitution mutagenesis has suggested functions for some of the side chains of the amino acids in this network in activation of the M 1 mAChR. Ala-substitutions of Leu 116 and Ser 120 simultaneously increased ACh affinity, raised basal activity, and enhanced signaling efficacy, suggesting that the intramolecular contacts made by these highly conserved residues help to stabilize the inactive ground state of the M 1 mAChR (3, 4). A similar role has been proposed for Phe 374 in TM 6 (7,22). Mutation of Ser 120 to histidine caused little change in agonist and antagonist binding. In contrast with mutation of S120A and S120C, 2 S120H strongly reduced signaling efficacy and maximum PI response. Double mutation of L116H-S120H and S120H-Y418H was inactive, but S120H-Y208H and S120H-F374H gave detectable PI response to ACh ( Table I). Substitution of the small side chain of Ser 120 by histidine may affect transition from the inactive to the active state, in which relative rotation and translation of helices are necessary, but without perturbation of the ground-state structure.
His substitution of the TM 7 residues Asn 414 and Tyr 418 (Table I) increased ACh affinity but essentially abolished G qmediated phosphoinositide signaling. These findings were confirmed by Ala-substitution. In the case of N414A, as reported for L116A, there was also a large reduction in receptor expression. 3 Asn 414 and Tyr 418 may resemble Leu 116 , Ser 120 , and Phe 374 in making intramolecular contacts that stabilize the inactive ground state of the receptor. However, the loss of signal implies that they are also important for the formation of the agonist-receptor-G q protein signaling complex. Thus, in contrast to residues which act as pure constraints, they may have an additional role. 3 Z.-L. Lu 3. Zn 2؉ binding to His-substitution mutants. a, changes in Zn 2ϩ binding affinity, relative to wild-type. b, cooperativity of Zn 2ϩ binding by substituted histidines. The values were calculated by the ratio of K Zn /(K Zn1 ϩ K Zn2 ϩ ...) minus 1, which corresponded to the contribution to the free energy (⌬⌬G o chelate ) arising from the chelate effect, ⌬⌬G o chelate ϭ ϪRT ln(K Zn /(K Zn1 ϩ K Zn2 ϩ ...)) (23), where K Zn is the association constant for Zn 2ϩ binding to a double or triple histidine mutant, K Zn1,2, are the association constants for Zn 2ϩ binding to the individual single histidine mutants, R is the gas constant (8.314 J/K/ mol), and T ϭ 298.15 K. Values are mean Ϯ standard error of three or more independent experiments.
In summary, several of the specific, conserved, interhelical contacts between TM 3, 6, and 7 of the rhodopsin-like 7-TM receptors which have been proposed from mutagenesis and modeling studies have been directly supported by the engineering of Zn 2ϩ binding sites in the M 1 mAChR. These contacts may be important in stabilizing the off-state of the receptor switch mechanism and be broken or rearranged during receptor activation. FIG. 4. Conserved interhelical constraint network demonstrated by Zn 2؉ Sites. The white triangle shows the interhelical triad whose histidine substitution gave the highest Zn 2ϩ affinity, indicating close proximity of the side chains of these amino acids. The green triangles show the triads whose histidine substitution also produced high-affinity binding sites for Zn 2ϩ . In each case, deletion of the side chain by Ala-substitution causes increases in agonist binding affinity.