Magnetic resonance studies of the binding of oligonucleotide substrates to mutants of staphylococcal nuclease

Woei‐Jer ‐J Chuang, apostolos G. Gittis, Albert S. Mildvan

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By a combination of NMR docking and model building, the substrate binding site on staphylococcal nuclease was found to accommodate a trinucleotide and to consist of three subsites, each interacting with a single nucleotidyl unit of DNA. Binding of the essential Ca2+ activator and substrate cleavage occur between subsites 1 and 2. Hence, catalytically productive binding would span subsites 1 and 2 while nonproductive binding would span subsites 2 and 3. Lys‐49 is near subsite 1, and Lys‐84 and Tyr‐115 interact with substrates at sub site 3 [Weber, D. J., Gittis, A. G., Mullen, G. P., Abeygunawardana, C., Lattman, E. E., Mildvan, A. S. Proteins 13:275–287, 1992]. The proposed locations of these subsites were independently tested by the effects of the K49A, K84A, and Y115A mutations of staphylococcal nuclease on the binding of Mn2+, Ca2+, and the dinucleotide and trinucleotide substrates, 5′‐pdTdA, dTdA, and dTdAdG. These three mutants have previously been shown to be fully active and to have CD and 2D NMR spectra very similar to those of the wild‐type enzyme (Chuang, W.‐J., Weber, D. J., Gittis, A. G., Mildvan, A. S. Proteins 17:36–48, 1993). All three mutant enzymes and their pdTdA and dTdA complexes (but not their dTdAdG complex) bind Mn2+ and Ca2+ more weakly than the wild‐type enzyme by factors ranging from 2 to 11. The presence of a terminal phosphate as in 5′‐pdTdA raises the affinity of the substrate for staphylococcal nuclease and its three mutants by two orders of magnitude and for the corresponding enzyme–metal complexes by three to four orders of magnitude, suggesting that the terminal phosphate is coordinated by the enzyme‐bound divalent cation. Such complexation would result in the nonproductive binding of 5′‐pdTdA at subsites 2 and 3. Accordingly, the K84A and Y115A mutations significantly weaken the binding of 5′‐pdTdA and its metal to staphylococcal nuclease by factors of 2.2 to 37.8, while the K49A mutation has much smaller or no effect. Such nonproductive binding explains the low activity of staphylococcal nuclease with small substrates, especially those With a terminal phosphate. Similarly, the K84A and Y115A mutations weaken the binding of dTdA and its metal complexes to the enzyme by factors of 3.4 to 13.1 while the K49A mutation has smaller effects indicating significant nonproductive binding of dTdA. The trinucleotide dTdAdG binds more tightly to wild‐type and mutant staphylococcal nuclease and to its metal complexes than does the dinucleotide dTdA by factors of 2.4 to 12.2, reflecting the occupancy of an additional subsite. Predominantly productive binding of dTdAdG is indicated by the 1.7− to 8.3−fold lower affinities of the K49A, K84A, and Y115A mutants for the trinucleotide and its metal complexes. The largest effects on dTdAdG binding are seen with the Y115A mutation presumably reflecting the dual role of Tyr‐115 both in donating a hydrogen bond to a phosphodiester oxygen between subsites 2 and 3 and in stacking onto the guanine base at subsite 3. © 1994 John Wiley & Sons, Inc.

Original languageEnglish
Pages (from-to)68-80
Number of pages13
JournalProteins: Structure, Function, and Bioinformatics
Issue number1
Publication statusPublished - 1994 Jan

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Biochemistry
  • Molecular Biology


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