Mapping protein-protein interactions with a library of tethered cutting reagents: the binding site of sigma 70 on Escherichia coli RNA polymerase.
Surface-exposed lysine amino groups and other reactive nucleophiles of the sigma 70 protein were conjugated with the cutting reagent iron (S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (FeBABE) via 2-iminothiolane (2IT) with low efficiency. The result is a library of sigma 70 conjugates, with an average of 1-2 cutting reagents tethered to any of a variety of sites (lysine, cysteine, etc.) on the surface of the protein. Model calculations indicate that the conjugates in this library should be capable of cutting nearby sites on the backbone of almost any protein or nucleic acid to which sigma 70 binds. Since cutting occurs only when the protein is bound, the cleaved sites indicate proximity; since only proximal sites are cleaved, interpretation of the results is straightforward. We used this library to map the periphery of the binding site on the core enzyme (alpha 2 beta beta') of Escherichia coli RNA polymerase. The beta subunit was cut primarily within its conserved regions C, D, Rif I, and G; additional sites were also cut between A and B and near conserved regions E and H. The cut sites within the beta' subunit were intensely clustered between residues 250-450, which include its conserved regions C and D, along with two additional cut sites in conserved regions A and G. No cut sites on the alpha subunit were observed. These results recapitulate and extend those obtained using FeBABE conjugates of seven strategically placed single-Cys sigma 70 mutants [Owens, J. T., Miyake, R., Murakami, K., Chmura, A. J., Fujita, N., Ishihama, A., and Meares, C. F. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 6021-6026]. This technique provides a straightforward, general approach to mapping protein interactions without mutagenesis.
Traviglia
S L
SL
Department of Chemistry, University of California, Davis 95616, USA.
Datwyler
S A
SA
Meares
C F
CF
D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007096:Imidoesters / Q000737:chemistry* / Q000378:metabolism; D007502:Iron Chelating Agents / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009942:Organometallic Compounds / Q000737:chemistry* / Q000378:metabolism; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods*; D012808:Sigma Factor / Q000737:chemistry* / Q000378:metabolism
D003432:Cross-Linking Reagents; D007096:Imidoesters; D007502:Iron Chelating Agents; D009942:Organometallic Compounds; D019151:Peptide Library; D012808:Sigma Factor; C005905:methyl 4-mercaptobutyrimidate; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases