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Mapping the Binding-Site Crevice of the D2 Receptor
JonathanA. Javitch Center for Molecular Recognition Departments of Psychiatry and Pharmacology College of Physicians and Surgeons Columbia University and N e w York State Psychiatric institute N e w York, N e w York 10032
Mapping the Binding-Site Crevice of the D2 Receptor The dopamine receptors, like the homologous receptors for biogenic amines, bind neurotransmitters present in the extracellular medium and couple this binding to the activation of intracellular G-proteins. The binding sites of these receptors are formed among their seven, mostly hydrophobic, membranespanning segments and are accessible to charged, water-soluble agonists, like dopamine. Thus, each of these binding sites is contained within a wateraccessible crevice, the binding-site crevice, extending from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed by residues that contact specific agonists and/or antagonists and other residues that may affect binding indirectly. In the homologous P2-adrenergic receptor, residues that contribute to binding have been identified in membrane-spanning segments, M3, M5, M6, and M7: Mutations of Asp 113, Ser 204, Ser 207, and Phe 290 altered binding and Trp 330 was affinity-labeled by an antagonist derivative. These five residues are identically conserved in all catecholamine receptors. In the dopamine D2 receptor, mutation of the residues that align with the first three, Asp 114, Ser 194, and Ser 197, also altered the binding of dopamine agonists and antagonists. Completely conserved residues, however, cannot account for the profound differences in binding specificities among the catecholamine receptors. Additional residues must contribute to binding, either directly or indirectly. To identify the residues that form the surface of the binding-site crevice in the human D2 receptor, we have used the substituted-cysteine accessibility method (SCAM) (1-5). Consecutive residues in the membrane-spanning segments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine-substitution mutant is near-normal, we assume that the structure of the mutant receptor, especially around the binding site, is similar to that of wild type and that the substituted cysteine lies in an orientation similar to that of the wildtype residue. In the membrane-spanning segments, the sulfhydryl of a cysteine can face either into the binding-site crevice, into the interior of the protein, or into the lipid bilayer; sulfhydryls facing into the binding-site crevice should react much faster with hydrophilic, lipophobic, and sulfhydryl-specific reagents. For such reagents, we use derivatives of methanethiosulfonate 412
Advances in Pharmacology, Volume 42 Copyright 0 1998 by Acadenuc Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
Mapping Binding-Site Crevice of D2 Receptor
4 13
(MTS): positively charged MTSethylammonium (MTSEA) and MTSethyltrimethylammonium (MTSET), and negatively charged MTSethylsulfonate (MTSES). These reagents are about th: same pize as dopamine, with maximum dimensions of approximately 10 A by 6 A. They form mixed disulfides with the cysteine sulfhydryl, covalently linking SCHzCH2X, where X is NH3+,N(CH3)3+,or SO,-. We use two criteria for identifying an engineeredcysteine as forming the surface of the binding-site crevice: (1)The reaction with an MTS reagent alters binding irreversibly and (2) this reaction is retarded by the presence of agonists or antagonists. A distinction between our approach and typical mutagenesis experiments is that we do not rely on the functional effects of a given mutation. The interpretation of the effects of typical mutagenesis experiments requires one to assume that functional changes, such as changes in binding affinity, caused by a mutation are only due to local effects at the site of the mutation and not due to nonlocal effects of the mutation on protein structure. The validity of this assumption is rarely proven for individual mutations. By contrast, it is unlikely that the protein segments lining the binding site would be grossly distorted in a dopamine-receptor mutant with near-normal binding properties. Thus, the engineered cysteine side-chain and the native side-chain are likely in close to the same position in the three-dimensional structure. We probe whether the engineered cysteine is accessible to our highly water soluble reagents, thereby determining whether it is on a water-accessible surface of the protein. An additional advantage of our approach is that we can determine whether a residue lines the binding-site crevice, even if mutation of the residue produces no functional change in the properties of the receptor. We previously found that antagonist binding to wild-type D2 receptor was irreversibly inhibited by MTSEA and MTSET and that Cys 118, in the third membrane-spanning segment (M3), was responsible for this sensitivity (2). Therefore, we used the mutant in which Cys 118 was replaced by serine (C118S), which is insensitive to MTS reagents, as the starting point for further mutation. In our initial application of the substituted-cysteine accessibility method to the D2 receptor (3), we found that 10 of 23 residues tested in the M3 segment were exposed in the binding-site crevice (Fig. 1A).From the pattern of exposure, we inferred that M 3 forms an a-helix, one side of which faces the binding-site crevice (Fig. 1B). Unlike the positively charged MTSEA and MTSET, the negatively charged MTSES did not react with cysteines substituted for residues more cytoplasmic than Val 111. This is consistent with a negative electrostatic potential in the binding-site crevice, in part due to the negative charge of Asp 114. However, MTSES reacted with the mutants D108C, I109C, F11OC, and V111C. At I109C, FllOC, and V111C, 10 mM MTSES inhibited binding nearly as much as 1 mM MTSET. Because 1 mM MTSET and 10 mM MTSES are equireactive with simple thiols in solution, the rates of reaction of MTSET and MTSES with the residues located near the extracellular end of M3 are similar; this indicates that the electrostatic potential near these residues is not as negative as it is below Val 111. Thirteen of 24 residues tested in the M5 segment of the D2 receptor were exposed in the binding-site crevice (4). Of the 13 exposed residues, 10 were
4 14
Jonathan A. javitch
A
B
MTSEA (2.5 mM) 1109c Fl10C v111c
M117C
wr (C118)
T119C A120C s121c
L123C N124C A127C I 128C s129c 113oc I '
0
25
H5 "
5
'
I
50
" "
I '
75
9
'
'
I
' "
100
Inhibi+bn (%)
FIGURE I
( A )The inhibition of specific ['H]YM-09151-2 binding to intact cells transiently
transfected with wild-type or mutant D2 receptors resulting from a 2-min application of 2.5 mM of MTSEA. The means and SEM are shown. The number of independent experiments for each mutant is shown next to the bars. Solid bars indicate mutants for which inhibition was significantly different ( p < .05) than C118S by one-way ANOVA. In C118S, Cys 126 is the only cysteine present in M3, but it is insensitive to the MTS reagents. Cys 126 is, thus, present in all the mutants. WT, wild-type; *,no detectable binding. ( B ) Helicalnet representationsofthe residues in and flanking the M3 segment of the dopamine D2 receptor, summarizing the effects of MTSEA on [3H]YM-09151-2binding. Reactiveresiduesare represented by squares, wherethe fill indicates the range of the second-order rate constants in M-'s-' for reaction with MTSEA: solid squares, k 2 20; hatched squares, 20 > k 2 10; striped squares, 10 > k 2 3; open square, 3 > k > 1. Small open circles indicate that MTSEA had no effect on binding. The solid circle indicates no binding after cysteine substitution. D108 and I109 are represented outside of the a-helix in the loop from M2. (Reprinted with permission from Neuron, vol. 14,825436,1995.)
consecutive. This pattern of exposure is inconsistent with the expectation that M.5, like M3, forms a fixed a-helix, one side of which is exposed in the bindingsite crevice. The exposed region of M5, which contains the serines likely to bind agonist, might loop out into the lumen of the binding-site crevice and be completely accessible to water and, thus, to MTSEA. Alternatively, the exposed region of M.5 might be embedded in the membrane and also in contact with other membrane-spanning segments. At any instant, only a limited set of residues might be exposed in the binding-site crevice; however, M.5 might move rapidly to expose different sets of residues. Nine of 26 residues tested in the M7 segment reacted with the MTS reagents and were protected from reaction by the antagonist sulpiride ( 5 ) . Again, the overall pattern of exposure is not consistent with a simple secondary structure of either a-helix or &strand. M7 contains the extremely highly conserved
Mapping Binding-Site Crevice of D2 Receptor
4 15
residues Am-Pro in the middle of the putative membrane-spanning segment. In soluble proteins, these residues have been observed to introduce kinks and twists in a-helices. The pattern of exposure of the cysteine substitution mutants to MTSEA can be explained if M7 is a kinked and twisted a-helix. References 1. Akabas, M. H., Stauffer, D. A., Xu, M., and Karlin, A. (1992). Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258, 307-310. 2. Javitch, J. A., Li, X., Kaback, J., and Karlin, A. (1994). A cysteine residue in the third membrane-spanning segment of the human dopamine D2 receptor is exposed in the bindingsite crevice. Proc. Nutl. Acad. Sci. U.S.A. 91, 10355-10359. 3. Javitch, J. A,, Fu, D., Chen, J., and Karlin, A. (1995). Mapping the binding-site crevice of the dopamine D2 dopamine receptor by the substituted-cysteine accessibility method. Neuron 14, 825-831. 4. Javitch, J. A., Fu, D., and Chen, J. (1995).Residues in the fifth membrane-spanning segment of the dopamine D2 receptor exposed in the binding-site crevice. Biochemistry 34, 1643316439. 5. Fu, D., Ballesteros, J. A., Weinstein, H., Chen, J., and Javitch, J. A. (1996). Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice. Biochemistry 3.5, 13278-11285.
Mapping the Binding-Site Crevice of the D2 Receptor The dopamine receptors, like the homologous receptors for biogenic amines, bind neurotransmitters present in the extracellular medium and couple this binding to the activation of intracellular G-proteins. The binding sites of these receptors are formed among their seven, mostly hydrophobic, membranespanning segments and are accessible to charged, water-soluble agonists, like dopamine. Thus, each of these binding sites is contained within a wateraccessible crevice, the binding-site crevice, extending from the extracellular surface of the receptor into the plane of the membrane. The surface of this crevice is formed by residues that contact specific agonists and/or antagonists and other residues that may affect binding indirectly. In the homologous P2-adrenergic receptor, residues that contribute to binding have been identified in membrane-spanning segments, M3, M5, M6, and M7: Mutations of Asp 113, Ser 204, Ser 207, and Phe 290 altered binding and Trp 330 was affinity-labeled by an antagonist derivative. These five residues are identically conserved in all catecholamine receptors. In the dopamine D2 receptor, mutation of the residues that align with the first three, Asp 114, Ser 194, and Ser 197, also altered the binding of dopamine agonists and antagonists. Completely conserved residues, however, cannot account for the profound differences in binding specificities among the catecholamine receptors. Additional residues must contribute to binding, either directly or indirectly. To identify the residues that form the surface of the binding-site crevice in the human D2 receptor, we have used the substituted-cysteine accessibility method (SCAM) (1-5). Consecutive residues in the membrane-spanning segments are mutated to cysteine, one at a time, and the mutant receptors are expressed in heterologous cells. If ligand binding to a cysteine-substitution mutant is near-normal, we assume that the structure of the mutant receptor, especially around the binding site, is similar to that of wild type and that the substituted cysteine lies in an orientation similar to that of the wildtype residue. In the membrane-spanning segments, the sulfhydryl of a cysteine can face either into the binding-site crevice, into the interior of the protein, or into the lipid bilayer; sulfhydryls facing into the binding-site crevice should react much faster with hydrophilic, lipophobic, and sulfhydryl-specific reagents. For such reagents, we use derivatives of methanethiosulfonate 412
Advances in Pharmacology, Volume 42 Copyright 0 1998 by Acadenuc Press. All rights of reproduction in any form reserved. 1054-3589/98 $25.00
Mapping Binding-Site Crevice of D2 Receptor
4 13
(MTS): positively charged MTSethylammonium (MTSEA) and MTSethyltrimethylammonium (MTSET), and negatively charged MTSethylsulfonate (MTSES). These reagents are about th: same pize as dopamine, with maximum dimensions of approximately 10 A by 6 A. They form mixed disulfides with the cysteine sulfhydryl, covalently linking SCHzCH2X, where X is NH3+,N(CH3)3+,or SO,-. We use two criteria for identifying an engineeredcysteine as forming the surface of the binding-site crevice: (1)The reaction with an MTS reagent alters binding irreversibly and (2) this reaction is retarded by the presence of agonists or antagonists. A distinction between our approach and typical mutagenesis experiments is that we do not rely on the functional effects of a given mutation. The interpretation of the effects of typical mutagenesis experiments requires one to assume that functional changes, such as changes in binding affinity, caused by a mutation are only due to local effects at the site of the mutation and not due to nonlocal effects of the mutation on protein structure. The validity of this assumption is rarely proven for individual mutations. By contrast, it is unlikely that the protein segments lining the binding site would be grossly distorted in a dopamine-receptor mutant with near-normal binding properties. Thus, the engineered cysteine side-chain and the native side-chain are likely in close to the same position in the three-dimensional structure. We probe whether the engineered cysteine is accessible to our highly water soluble reagents, thereby determining whether it is on a water-accessible surface of the protein. An additional advantage of our approach is that we can determine whether a residue lines the binding-site crevice, even if mutation of the residue produces no functional change in the properties of the receptor. We previously found that antagonist binding to wild-type D2 receptor was irreversibly inhibited by MTSEA and MTSET and that Cys 118, in the third membrane-spanning segment (M3), was responsible for this sensitivity (2). Therefore, we used the mutant in which Cys 118 was replaced by serine (C118S), which is insensitive to MTS reagents, as the starting point for further mutation. In our initial application of the substituted-cysteine accessibility method to the D2 receptor (3), we found that 10 of 23 residues tested in the M3 segment were exposed in the binding-site crevice (Fig. 1A).From the pattern of exposure, we inferred that M 3 forms an a-helix, one side of which faces the binding-site crevice (Fig. 1B). Unlike the positively charged MTSEA and MTSET, the negatively charged MTSES did not react with cysteines substituted for residues more cytoplasmic than Val 111. This is consistent with a negative electrostatic potential in the binding-site crevice, in part due to the negative charge of Asp 114. However, MTSES reacted with the mutants D108C, I109C, F11OC, and V111C. At I109C, FllOC, and V111C, 10 mM MTSES inhibited binding nearly as much as 1 mM MTSET. Because 1 mM MTSET and 10 mM MTSES are equireactive with simple thiols in solution, the rates of reaction of MTSET and MTSES with the residues located near the extracellular end of M3 are similar; this indicates that the electrostatic potential near these residues is not as negative as it is below Val 111. Thirteen of 24 residues tested in the M5 segment of the D2 receptor were exposed in the binding-site crevice (4). Of the 13 exposed residues, 10 were
4 14
Jonathan A. javitch
A
B
MTSEA (2.5 mM) 1109c Fl10C v111c
M117C
wr (C118)
T119C A120C s121c
L123C N124C A127C I 128C s129c 113oc I '
0
25
H5 "
5
'
I
50
" "
I '
75
9
'
'
I
' "
100
Inhibi+bn (%)
FIGURE I
( A )The inhibition of specific ['H]YM-09151-2 binding to intact cells transiently
transfected with wild-type or mutant D2 receptors resulting from a 2-min application of 2.5 mM of MTSEA. The means and SEM are shown. The number of independent experiments for each mutant is shown next to the bars. Solid bars indicate mutants for which inhibition was significantly different ( p < .05) than C118S by one-way ANOVA. In C118S, Cys 126 is the only cysteine present in M3, but it is insensitive to the MTS reagents. Cys 126 is, thus, present in all the mutants. WT, wild-type; *,no detectable binding. ( B ) Helicalnet representationsofthe residues in and flanking the M3 segment of the dopamine D2 receptor, summarizing the effects of MTSEA on [3H]YM-09151-2binding. Reactiveresiduesare represented by squares, wherethe fill indicates the range of the second-order rate constants in M-'s-' for reaction with MTSEA: solid squares, k 2 20; hatched squares, 20 > k 2 10; striped squares, 10 > k 2 3; open square, 3 > k > 1. Small open circles indicate that MTSEA had no effect on binding. The solid circle indicates no binding after cysteine substitution. D108 and I109 are represented outside of the a-helix in the loop from M2. (Reprinted with permission from Neuron, vol. 14,825436,1995.)
consecutive. This pattern of exposure is inconsistent with the expectation that M.5, like M3, forms a fixed a-helix, one side of which is exposed in the bindingsite crevice. The exposed region of M5, which contains the serines likely to bind agonist, might loop out into the lumen of the binding-site crevice and be completely accessible to water and, thus, to MTSEA. Alternatively, the exposed region of M.5 might be embedded in the membrane and also in contact with other membrane-spanning segments. At any instant, only a limited set of residues might be exposed in the binding-site crevice; however, M.5 might move rapidly to expose different sets of residues. Nine of 26 residues tested in the M7 segment reacted with the MTS reagents and were protected from reaction by the antagonist sulpiride ( 5 ) . Again, the overall pattern of exposure is not consistent with a simple secondary structure of either a-helix or &strand. M7 contains the extremely highly conserved
Mapping Binding-Site Crevice of D2 Receptor
4 15
residues Am-Pro in the middle of the putative membrane-spanning segment. In soluble proteins, these residues have been observed to introduce kinks and twists in a-helices. The pattern of exposure of the cysteine substitution mutants to MTSEA can be explained if M7 is a kinked and twisted a-helix. References 1. Akabas, M. H., Stauffer, D. A., Xu, M., and Karlin, A. (1992). Acetylcholine receptor channel structure probed in cysteine-substitution mutants. Science 258, 307-310. 2. Javitch, J. A., Li, X., Kaback, J., and Karlin, A. (1994). A cysteine residue in the third membrane-spanning segment of the human dopamine D2 receptor is exposed in the bindingsite crevice. Proc. Nutl. Acad. Sci. U.S.A. 91, 10355-10359. 3. Javitch, J. A,, Fu, D., Chen, J., and Karlin, A. (1995). Mapping the binding-site crevice of the dopamine D2 dopamine receptor by the substituted-cysteine accessibility method. Neuron 14, 825-831. 4. Javitch, J. A., Fu, D., and Chen, J. (1995).Residues in the fifth membrane-spanning segment of the dopamine D2 receptor exposed in the binding-site crevice. Biochemistry 34, 1643316439. 5. Fu, D., Ballesteros, J. A., Weinstein, H., Chen, J., and Javitch, J. A. (1996). Residues in the seventh membrane-spanning segment of the dopamine D2 receptor accessible in the binding-site crevice. Biochemistry 3.5, 13278-11285.