Recognition of specific and nonspecific DNA by human lactoferrin

Abstract

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.

How human IgGs against DNA recognize oligonucleotides and DNA

“…In contrast to DNA polymerases, uracil‐DNA glycosylase (Vinogradova et al, ) AP endonuclease, and topoisomerase I (Topo I), but similarly to Escherichia coli 8‐oxoguanine DNA glycosylase (Fpg), human 8‐oxoguanine DNA glycosylase (OGG1), and Eco RI endonuclease, the affinity of human lactoferrin for d(pC) n , d(pT) n , and d(pA) n homo‐ODNs did not depend on the bases and their relative hydrophobicity; it depended only on the number of internucleoside phosphate groups . HIV‐1 integrase was the first enzyme, in which the affinity for nonspecific d(pC) n , d(pT) n , and d(pA) n was dependent on the bases but did not follow their relative hydrophobicity .…”

“…d(pA) 8 2.2 × 10 −8 6.7 × 10 −7 d(pC) 6 1.0 × 10 −8 7.9 × 10 −7 d(pT) 6 1.0 × 10 −9 2.7 × 10 −7 d(pA) 9 2.2 × 10 −8 4.5 × 10 −7 d(pC) 8 3.7 × 10 −9 6.6 × 10 −7 d(pT) 8 1.1 × 10 −8 2.3 × 10 −7 d(pA) 10 2.1 × 10 −8 3.5 × 10 −7 d(pC) 9 3.5 × 10 −9 4.6 × 10 −7 d(pT) 10 1.4 × 10 −8 2.4 × 10 −7 d(pA) 12 1.9 × 10 −8 3.4 × 10 −7 d(pC) 10 3.2 × 10 −9 3.9 × 10 −7 d(pT) 11 1.4 × 10 −8 2.9 × 10 −7 d(pA) 13 1.9 × 10 −8 3.1 × 10 −7 d(pC) 12 3.1 × 10 −9 3.8 × 10 −7 d(pT) 16 1.3 × 10 −8 2.0 × 10 −7 d(pA) 15 1.9 × 10 −8 2.9 × 10 −7 d(pC) 14 2.9 × 10 −9 3.6 × 10 −7 d(pT) 24 1.3 × 10 −8 1.6 × 10 −7 d(pA) 16 1.9 × 10 −8 2.3 × 10 −7 d(pC) 16 3.0 × 10 In contrast to DNA polymerases, [10][11][12][13] uracil-DNA glycosylase (Vinogradova et al, 25 ) AP endonuclease, 16 and topoisomerase I (Topo I), 19,20 but similarly to Escherichia coli 8-oxoguanine DNA glycosylase (Fpg), 23,27 human 8-oxoguanine DNA glycosylase (OGG1), 24 and EcoRI endonuclease, 15 the affinity of human lactoferrin for d(pC) n , d(pT) n , and d(pA) n homo-ODNs did not depend on the bases and their relative hydrophobicity; it depended only on the number of internucleoside phosphate groups. 22 HIV-1 integrase was the first enzyme, in which the affinity for nonspecific d(pC) n , d(pT) n , and d(pA) n was dependent on the bases but did not follow their relative hydrophobicity. 21 The affinity of d(A) n and d(C) n (having minimal and maximal relative hydrophobicity) was similar, while the affinity of d(T) n was significantly lower.…”

“…[10][11][12] To evaluate the relative contributions of individual DNA elements to the enzyme affinity for long DNA, a previously described approach, stepwise increase in ligand complexity (or SILC) was developed (for details, see Supporting Information). Using the SILC approach, we have studied many DNA-dependent enzymes, including those not specific and specific for DNA structure or sequences [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] (Vinogradova et al,25 ) (for details, see Supporting Information). The data are reviewed by Nevinsky.…”

How human IgGs against myelin basic protein (MBP) recognize oligopeptides and MBP

“… To evaluate the relative contributions of individual DNA elements to the enzyme affinity for long polymeric and oligomeric ligands, a new approach, stepwise increase in ligand complexity (SILC), was developed . Using the SILC, we have analyzed the mechanisms of recognition of DNA by number of DNA‐dependent enzymes: not specific for DNA sequence and structure such as Escherichia coli RecA, specific for DNA structure but not sequence‐dependent such as DNA polymerases of eukaryotes, prokaryotes, viruses, and archaea and human DNA ligase I; specific for DNA containing damages such as human uracil DNA glycosylase, E. coli Fpg and human 8‐oxoguanine DNA glycosylases (OGG1), human apurinic/apyrimidinic endonuclease (AP endonuclease), as well as specific for DNA sequence such as human HIV integrase, topoisomerase I (Topo I), Eco RI restriction endonuclease, and human lactoferrin (LF) . It was shown that complex formation including formation of contacts between specific sequences in all these enzymes cannot provide for either substrate specificity or high enzyme affinity for DNA.…”

“…human lactoferrin (LF). 20 It was shown that complex formation including formation of contacts between specific sequences in all these enzymes cannot provide for either substrate specificity or high enzyme affinity for DNA. All these enzymes recognize DNA by the formation of multiple additive contacts with all DNA nucleotide units covered by the protein globule (7-20 units depending on the enzyme).…”