Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli.
Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity. In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro. L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe. The cloned gene was overexpressed and purified as a his-tag fusion protein in E. coli. Radioligand binding experiments, using [gamma-35S]ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity. Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates. Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues. Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of [gamma-35S]ATP at subclinical levels. Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts.
Avliyakulov
N K
NK
Department of Pharmacology, Faculty of Health Sciences, University of Linköping, Sweden.
Lukes
J
J
Kajava
A V
AV
Liedberg
B
B
Lundström
I
I
Svensson
S P
SP
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D006160:Guanosine Triphosphate / Q000378:metabolism; D016830:Kinetoplastida / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013498:Suramin / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014544:Uridine Triphosphate / Q000378:metabolism
D009711:Nucleotides; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; D014344:Trypanocidal Agents; D013498:Suramin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D014544:Uridine Triphosphate