Recognition of specific and nonspecific DNA by human lactoferrin.
The general principles of recognition of nucleic acids by proteins are among the most exciting problems of molecular biology. Human lactoferrin (LF) is a remarkable protein possessing many independent biological functions, including interaction with DNA. In human milk, LF is a major DNase featuring two DNA-binding sites with different affinities for DNA. The mechanism of DNA recognition by LF was studied here for the first time. Electrophoretic mobility shift assay and fluorescence measurements were used to probe for interactions of the high-affinity DNA-binding site of LF with a series of model-specific and nonspecific DNA ligands, and the structural determinants of DNA recognition by LF were characterized quantitatively. The minimal ligands for this binding site were orthophosphate (K(i)  = 5 mM), deoxyribose 5'-phosphate (K(i)  = 3 mM), and different dNMPs (K(i)  = 0.56-1.6 mM). LF interacted additionally with 9-12 nucleotides or nucleotide pairs of single- and double-stranded ribo- and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleoside phosphate groups. Such nonspecific interactions of LF with noncognate single- and double-stranded d(pN)(10) provided ~6 to ~7.5 orders of magnitude of the enzyme affinity for any DNA. This corresponds to the Gibbs free energy of binding (ΔG(0)) of -8.5 to -10.0 kcal/mol. Formation of specific contacts between the LF and its cognate DNA results in an increase of the DNA affinity for the enzyme by approximately 1 order of magnitude (K(d)  = 10 nM; ΔG(0)  ≈ -11.1 kcal/mol). A general function for the LF affinity for nonspecific d(pN)(n) of different sequences and lengths was obtained, giving the K(d) values comparable with the experimentally measured ones. A thermodynamic model was constructed to describe the interactions of LF with DNA.
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentiev Ave., Novosibirsk 630090, Russia.
Tat'yana A
TA
Guschina
Svetlana E
SE
Soboleva
Georgy A
GA
Nevinsky
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007781:Lactoferrin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D008024:Ligands; D009841:Oligonucleotides; D004247:DNA; D007781:Lactoferrin