Details of the nucleic acid binding site of T4 gene 32 protein revealed by proteolysis and DNA Tm depression methods.
The affinities and location of oligonucleotides bound to intact and truncated bacteriophage T4 gene 32 protein have been elucidated by two independent and sensitive methods. The nucleic acid binding site is located within the core domain of 32 protein, residues 22-253. Oligonucleotides protect the core domain against proteolysis catalyzed by mammalian endoproteinase Arg-C. Of the three cleavage sites, Arg111, within the internal "LAST" ((Lys/Arg)3(Ser/Thr)2) motif, is selectively protected. We have previously suggested that these LAST residues, Lys-Arg-Lys-Thr-Ser, residues 110-114, are involved in nucleic acid binding, and our results are also consistent with crystallographic studies. The inhibitory effects of oligonucleotides on the kinetics of core domain proteolysis were used to quantify binding affinities. In addition, affinities of oligonucleotides for both core domain and intact protein were obtained from their effect on the Tm-depressing activities of these proteins. For both core and intact protein, the degree of affinity increases with oligonucleotide length. The presence of a 5' terminal phosphate increases the affinity two- to fourfold. Placement of methylphosphonodiester (uncharged) linkages at alternating linkages vastly lowers binding affinity for the intact protein and core domain. We conclude that at least two and likely three adjacent phosphodiester linkages are a minimal requirement for binding, further defining the electrostatic component of the interaction. The length-dependence of binding affinity suggests that additional interactions, both ionic and non-ionic, likely occur with longer oligonucleotides.
Wu
M
M
Department of Chemistry and Biochemistry, University of Maryland, Baltimore, MD, 21250, USA.
Flynn
E K
EK
Karpel
R L
RL
D017122:Bacteriophage T4 / Q000378:metabolism; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004563:Electrochemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; C044499:submandibular proteinase A; D012697:Serine Endopeptidases