A strained DNA binding helix is conserved for site recognition, folding nucleation, and conformational modulation

Abstract

Nucleic acid recognition is often mediated by alpha-helices or disordered regions that fold into alpha-helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical alpha-helix is stabilized at the N-terminus, while the PII forms at the C-terminus half of the peptide. Re-examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N-terminus half is a canonical alpha-helix, while the C-terminal half adopts a 3(10) helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10(4)-fold lower affinity, and 500-fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained alpha-helix conformation, "presented" by this unique architecture. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 432-443, 2009.

Indirect DNA Readout on the Protein Side: Coupling between Histidine Protonation, Global Structural Cooperativity, Dynamics, and DNA Binding of the Human Papillomavirus Type 16 E2C Domain

“…Quaternary interactions still show less protection than tertiary hydrogen bonds, and the 3 10 segment of the DNA recognition helix exchanges faster than the α-helix segment, as expected from its malleability. 41 The general slowdown of exchange is in agreement with the reduction of motions in the DNA binding helix and the β-barrel observed upon DNA binding (Figs. 5c and 6b).…”

“…Most E2C backbone amide nitrogens titrate with apparent pK a values that are almost identical with those determined for one of the five histidine residues (Table 3 and Wetzler et al 41 ). We could not measure the influence of His322 and His326 protonation on the structure of the β2-β3 loop because most residues in this region cannot be monitored above pH 5.…”

“…However, the effect of this change on histidine protonation is only local. 41 On the contrary, residues that contact the DNA (evidenced by black circles in the figure) show an almost identical chemical shift, indicating that the protein-DNA interface conformation is invariant between pH 5.0 and neutral pH.…”

“…28,29 Helix 1 is best described as a strained bipartite helix, with a canonical α-helix N-terminal half and a 3 10 helix C-terminal half. 41 The dimeric topology of E2C is unusual, with the DNA binding domain of the Epstein-Barr nuclear antigen 1 being the only known structural analog. 42 In spite of the structural similarity between these two viral DNA binding domains, their amino acid sequences and DNA binding specificities show no detectable homology.…”

Experimental snapshots of a protein-DNA binding landscape

“…The free E2C domain is stable (27) yet highly dynamic, with less than half of backbone amide protons showing significant protection from exchange with the solvent (24), while most backbone amide protons are shielded from exchange in DNA-bound E2C (24). Moreover, the conformational diversity of the DNAbinding helix is restricted upon binding (35) and internal conformational rearrangements in the homodimeric β-barrel core take place (24). We propose that these conformational rearrangements involve internal interactions of E2C and contribute to the large free energy barrier for conversion of the nonnative I late into the final complex and its major enthalpic component (4).…”