ABSTRACT
Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.
Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/chemistry , Evolution, Molecular , GTP-Binding Proteins/chemistry , Guanosine Triphosphate/chemistry , Monomeric GTP-Binding Proteins/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Guanosine Triphosphate/genetics , Guanosine Triphosphate/metabolism , Humans , Hydrolysis , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , Protein Structure, Tertiary , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Substrate SpecificityABSTRACT
Nuclear accumulation of the complex between beta-catenin and proteins of the T-cell factor (Tcf) family is a hallmark of many cancers. Targeting this interaction for drug development is complicated by the fact that E-cadherin and adenomatous polyposis coli (APC) bind to overlapping sites on beta-catenin. Inhibiting their interactions might actually promote tumor growth. To identify selective beta-catenin binding hot spots of Tcf4, E-cadherin, and APC, array technology with peptides of up to 53 amino acids length was used. Interactions were monitored by a quantitative fluorescent readout, which was shown to represent a monitor of true equilibrium binding constants. We identified minimal binding motifs in the beta-catenin ligands and showed that most of the 15-mer and 20-mer repeats of APC did not interact, at least when non-phosphorylated, and defined a consensus binding motif also present in APC. We confirmed previously found hot spots and identified new ones. The method allowed us to locate a hydrophobic pocket that was relevant for the Tcf, but not the E-cadherin interaction, and would thus constitute an ideal drug target site.
Subject(s)
Cadherins/metabolism , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/metabolism , Peptides/chemical synthesis , Protein Array Analysis , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Binding Sites , Cadherins/chemistry , Combinatorial Chemistry Techniques , Cytoskeletal Proteins/chemistry , DNA-Binding Proteins/chemistry , Drug Delivery Systems , Humans , Ligands , Mice , Models, Molecular , Peptides/metabolism , Protein Interaction Mapping/methods , TCF Transcription Factors , Trans-Activators/chemistry , Transcription Factor 7-Like 2 Protein , Transcription Factors/chemistry , beta CateninABSTRACT
A practical and convenient method for the synthesis of acid- and base-sensitive GTP analogues carrying a further substituent at the terminal phosphate has been developed. Key to the successful synthesis of these potential ligands of the Ras protein is the use of Pd0-sensitive allyl protecting groups in a one-pot synthesis that avoids evaporation steps. Initial biochemical analysis of a representative compound revealed that such GTP analogues can bind to Ras and might open up the possibility of developing small molecules that can act as deactivators of oncogenic Ras.
