STRUCTURE OF THE NICOTINIC RECEPTOR ACETYLCHOLINE-BINDING SITE - IDENTIFICATION OF ACIDIC RESIDUES IN THE DELTA-SUBUNIT WITHIN 0.9-NM OF THE ALPHA-SUBUNIT-BINDING SITE DISULFIDE
C. Czajkowski et A. Karlin, STRUCTURE OF THE NICOTINIC RECEPTOR ACETYLCHOLINE-BINDING SITE - IDENTIFICATION OF ACIDIC RESIDUES IN THE DELTA-SUBUNIT WITHIN 0.9-NM OF THE ALPHA-SUBUNIT-BINDING SITE DISULFIDE, The Journal of biological chemistry, 270(7), 1995, pp. 3160-3164
In the nicotinic receptor, the quaternary ammonium group of acetylchol
ine (ACh) binds to a negative subsite at most 1 nm from a readily redu
cible disulfide formed between alpha-subunit residues Cys(192) and Cys
(193). The cross-linker S-(2-[H-3]glycylamidoethyl)dithio-2-pyridine f
ormed a disulfide bond with reduced alpha Cys(192)/Cys(193) and an ami
de bond with an acidic residue in the delta subunit (Czajkowski, C., a
nd Karlin, A (1991) J. Biol. Chem. 266, 22603-22612). The fully extend
ed cross-linking moiety -NRCH(2)CONHCH(2)CH(2)S- is 0.9 nm long. After
the disulfide bond linking -NRCH(2)CONHCH(2)CH(2)S- to the alpha subu
nit was reduced, -NHCH2CONHCH2CH2SH remained linked to the delta subun
it by an amide bond. The delta subunit was cleaved at Met residues, an
d the radioactive fragments were isolated and sequenced by automated E
dman degradation. Additionally, the isolated radioactive fragments wer
e further cleaved at Trp residues and sequenced. Peaks of release of r
adioactivity were obtained in the sequencing cycles corresponding to d
elta Asp(165), delta Asp(180) and delta Glu(182). The mutation of delt
a Asp(180) to Asn decreased the affinity of the receptor for ACh 100-f
old, whereas the mutation of either delta Asp(165) delta Glu(182), Or
8 Other acidic residues in the same region of delta decreased the affi
nity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A. (
1993) Proc. Natl. Acad. Sci. U. S. A 90, 6285-6289). Because delta Asp
(180) both contributes to ACh binding and is suitably close to the bin
ding site disulfide, it is likely to be part of the ACh-binding site f
ormed in the interface between the alpha and the delta subunits.