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Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity.

ABSTRACT: Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase. Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB). The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue. The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S. The renatured mixture was treated with DTNB and separated on an NAD(+)-agarose column to which only C320S subunits bind tightly. Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry. A fraction highly enriched in the 1:5 hybrid was identified. Homohexamers (C320S with 40 mM glutamate and 1 mM NAD(+) at pH 8.8, or K89L/A163G/S380A with 70 mM norleucine and 1 mM NAD(+) at pH 8.5) showed allosteric activation; succinate activated C320S approx. 50-fold (EC(50)=70 mM, h=2.4), and glutarate gave approx. 30-fold activation (EC(50)=35 mM, h=2.3). For the triple mutant, corresponding values were 80 mM and 2.2 for succinate, and 75 mM and 1.7 for glutarate, but maximal activation was only about 2-fold. In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer. A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites. On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine. With glutamate at pH 8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer. The "foreign" subunit evidently impedes inter-subunit communication to some extent.

SUBMITTER: Goyal A

PROVIDER: S-EPMC1222269 | biostudies-other | 2001 Dec

REPOSITORIES: biostudies-other

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Project description:Homomeric ?7 nicotinic acetylcholine receptors (nAChR) have an intrinsically low probability of opening that can be overcome by ?7-selective positive allosteric modulators (PAMs), which bind at a site involving the second transmembrane domain (TM2). Mutation of a methionine that is unique to ?7 at the 15' position of TM2 to leucine, the residue in most other nAChR subunits, largely eliminates the activity of such PAMs. We tested the effect of the reverse mutation (L15'M) in heteromeric nAChR receptors containing ?4 and ?2, which are the nAChR subunits that are most abundant in the brain. Receptors containing these mutations were found to be strongly potentiated by the ?7 PAM 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS) but insensitive to the alternative PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea. The presence of the mutation in the ?2 subunit was necessary and sufficient for TQS sensitivity. The primary effect of the mutation in the ?4 subunit was to reduce responses to acetylcholine applied alone. Sensitivity to TQS required only a single mutant ? subunit, regardless of the position of the mutant ? subunit within the pentameric complex. Similar results were obtained when ?2L15'M was coexpressed with ?2 or ?3 and when the L15'M mutation was placed in ?4 and coexpressed with ?2, ?3, or ?4. Functional receptors were not observed when ?1L15'M subunits were coexpressed with other muscle nAChR subunits. The unique structure-activity relationship of PAMs and the ?4?2L15'M receptor compared with ?7 and the availability of high-resolution ?4?2 structures may provide new insights into the fundamental mechanisms of nAChR allosteric potentiation. SIGNIFICANCE STATEMENT: Heteromeric neuronal nAChRs have a relatively high initial probability of channel activation compared to receptors that are homomers of ?7 subunits but are insensitive to PAMs, which greatly increase the open probability of ?7 receptors. These features of heteromeric nAChR can be reversed by mutation of a single residue present in all neuronal heteromeric nAChR subunits to the sequence found in ?7. Specifically, the mutation of the TM2 15' leucine to methionine in ? subunits reduces heteromeric receptor channel activation, while the same mutation in neuronal ? subunits allows heteromeric receptors to respond to select ?7 PAMs. The results indicate a key role for this residue in the functional differences in the two main classes of neuronal nAChRs.

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Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity.

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