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Ligand Interaction Scan: a general method for engineering small molecule ligand‐sensitive protein alleles
Author(s) -
Liscovitch Moti,
Erster Oran,
Eisenstein Miri
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb17-d
Subject(s) - ligand (biochemistry) , small molecule , chemistry , mutant , in silico , protein engineering , biophysics , mutagenesis , target protein , biochemistry , biology , receptor , gene , enzyme
Chemical genetic methods that harness the power of small molecule pharmacology for evaluating the cellular functions of certain protein classes were recently developed. Here we describe a novel, general and simple procedure for engineering small molecule ligand‐regulated forms of any protein. The ligand interaction scan method involves insertional mutagenesis of a given protein by a chemical‐genetic switch, comprising a genetically‐encoded peptide module that binds with high affinity to a small molecule ligand. The insertion position is selected empirically to confer ligand‐dependent modulation of activity. To demonstrate the feasibility of the new method we applied it to the TEM‐1 beta‐lactamase antibiotics resistance gene. A tetracysteine (4C) hexapeptide and a cell‐permeable biarsenical fluorescein derivative (FlAsH) were utilized as peptide insert and ligand, respectively. We generated several ligand‐sensitive mutants of TEM‐1, of which two (4C‐97 and 4C‐200) are inhibited by FlAsH, whereas a third (4C‐215) is stimulated by the ligand. In‐silico molecular modeling of TEM‐1 4C‐215 suggests that FlAsH may act by stabilizing an open conformation of 4C‐215 in which the enzyme active site is exposed. The method was successfully applied also to a mammalian protein kinase. Our results demonstrate that drug‐sensitive alleles of TEM‐1 can be generated by ligand interaction scan and suggest that the method may be applied to any protein given an appropriate activity assay. Ligand‐regulated mutants may then be expressed in cells or inserted genomically, wherein they can be regulated by ligand administration. Because of its simplicity and generality the ligand interaction scan method may complement other genetic and chemical genetic methods for analysis of protein function and drug target validation.