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Mutations of recombinant rat liver fatty acid-binding protein at residues 102 and 122 alter its structural integrity and affinity for physiological ligands.

ABSTRACT: Rat liver fatty acid-binding protein (FABP) is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. This property means that the liver protein is functionally different from other FABPs from intestine, muscle and adipose tissue that have a more restricted ligand specificity and stoichiometry. The availability of crystal structures for the latter proteins has highlighted the importance of two arginine residues that are involved in the binding of the fatty acid carboxylate. Only one of these arginine residues, arginine-122, is conserved in liver FABP, whereas the other arginine, at position 102, is replaced by a threonine. In order to gain further insight into the nature of ligand interactions with liver FABP these key residues (102 and 122) have been changed by site-directed mutagenesis. The results with an R122Q mutant highlight the critical role of this arginine in determining ligand affinity, while similar but less dramatic effects were observed with the T102Q mutant. The double mutant T102Q/R122Q was expressed but had lost the ability to bind fluorescent ligands. It is concluded that Arg-122 plays a role in accommodating the carboxylate group of at least one fatty acid. It is proposed that physiological ligands with more bulky headgroups, such as lysophospholipids, acyl-CoA and mono-olein, bind with the headgroups in a solvent-exposed location near the portal region of the protein. The portal region is suggested to be more flexible in the mutants (R122Q and to a lesser extent T102Q). The net result is that the ligand specificity of the R122Q mutant changes to that of a protein with enhanced affinity for acyl-CoA, lysophospholipids and mono-olein.

SUBMITTER: Thumser AE

PROVIDER: S-EPMC1217148 | biostudies-other | 1996 Mar

REPOSITORIES: biostudies-other

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Project description:Etomidate and propofol are potent general anesthetics that act via GABAA receptor allosteric co-agonist sites located at transmembrane ?+/?- inter-subunit interfaces. Early experiments in heteromeric receptors identified ?N265 (M2-15') on ?2 and ?3 subunits as an important determinant of sensitivity to these drugs. Mechanistic analyses suggest that substitution with serine, the ?1 residue at this position, primarily reduces etomidate efficacy, while mutation to methionine eliminates etomidate sensitivity and might prevent drug binding. However, the ?N265 residue has not been photolabeled with analogs of either etomidate or propofol. Furthermore, substituted cysteine modification studies find no propofol protection at this locus, while etomidate protection has not been tested. Thus, evidence of contact between ?N265 and potent anesthetics is lacking and it remains uncertain how mutations alter drug sensitivity. In the current study, we first applied heterologous ?1?2N265C?2L receptor expression in Xenopus oocytes, thiol-specific aqueous probe modification, and voltage-clamp electrophysiology to test whether etomidate inhibits probe reactions at the ?-265 sidechain. Using up to 300 µM etomidate, we found both an absence of etomidate effects on ?1?2N265C?2L receptor activity and no inhibition of thiol modification. To gain further insight into anesthetic insensitive ?N265M mutants, we applied indirect structure-function strategies, exploiting second mutations in ?1?2/3?2L GABAA receptors. Using ?1M236C as a modifiable and anesthetic-protectable site occupancy reporter in ?+/?- interfaces, we found that ?N265M reduced apparent anesthetic affinity for receptors in both resting and GABA-activated states. ?N265M also impaired the transduction of gating effects associated with ?1M236W, a mutation that mimics ?+/?- anesthetic site occupancy. Our results show that ?N265M mutations dramatically reduce the efficacy/transduction of anesthetics bound in ?+/?- sites, and also significantly reduce anesthetic affinity for resting state receptors. These findings are consistent with a role for ?N265 in anesthetic binding within the ?+/?- transmembrane sites.

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Mutations of recombinant rat liver fatty acid-binding protein at residues 102 and 122 alter its structural integrity and affinity for physiological ligands.

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