STRUCTURAL CHARACTERISTICS OF THE NUCLEOTIDE-BINDING SITE OF ESCHERICHIA-COLI PRIMARY REPLICATIVE HELICASE DNAB PROTEIN - STUDIES WITH RIBOSE AND BASE-MODIFIED FLUORESCENT NUCLEOTIDE ANALOGS

Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichi a coil primary replicative helicase DnaB protein have been studied, us ing the base-modified fluorescent nucleotide analog 1, N-6-ethenoadeno sine diphosphate (epsilon ADP) and the ribose-modified fluorescent ana logs 3'(2')-O-(N-methylantraniloyl)adenosine 5'-diphosphate (MANT-ADP) , 3'-O-(N-methylantraniloyl) deoxyadenosine 5'-diphosphate (MANT-dADP) , 3'-O-(N-methylantraniloyl) deoxyadenosine 5'-triphosphate (MANT-dATP ), and 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-diphosphate (TNP-A DP). The obtained data indicate contrasting differences between these two regions. Binding of epsilon ADP to the DnaB helicase causes only s imilar to 21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum. The fluorescence of bound eps ilon ADP is characterized by a single lifetime of 24.2 +/- 0.6 ns, onl y slightly shorter than the fluorescent lifetime of the free epsilon A DP in solution (25.5 +/- 0.6 ns)l Solute-quenching studies of bound ep silon ADP, using different quenchers, acrylamide, I-, and Tl+, indicat e limited accessibility of ethenoadenosine to the solvent. These resul ts strongly suggest that the base-binding region of the DnaB nucleotid e-binding site is located in the polar cleft on the enzyme's surface. Moreover, the limiting emission anisotropy of bound epsilon ADP is 0.2 1 +/- 0.02, compared to the anisotropy of 0.3 of completely immobilize d epsilon ADP at the same excitation wavelength (lambda(ex) - 325 nm, lambda(em) = 410 nm), indicating that the adenine preserves substantia l mobility when bound in the base-binding site. In contrast, fluoresce nce intensity at the emission maximum of TNP-ADP and MANT-ADP, which h as modifying groups attached to the 2' and/or 3' oxygens of the ribose , increases upon binding to DnaB by factors of similar to 4.7 (lambda( ex) = 408 nm) and similar to 2.6 (lambda(ex) = 356 nm), respectively. Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-sh ifted by similar to 11 nm and that of MANT-ADP by similar to 12 nm. Co mparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity. Solu te quenching studies of MANT-ADP fluorescence, using acrylamide, I-, a nd Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB. Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or poc ket, with very limited, if any, contact with the solvent. Moreover, fl uorescence anisotropy of bound TNP-ADP and MANT-ADP is 0.32 +/- 0.02 a nd 0.33 +/- 0.02, respectively. These values are very close to the fun damental anisotropies of TNP-ADP and MANT-ADP, indicating that the flu orophores attached to the ribose have very restricted motional freedom . Fluorescence of MANT-ADP free in solution decays with a nearly homog enous single lifetime of 3.9 +/- 0.2 ns. However, upon binding, the em ission is characterized by two components (tau(1) = 13.1 +/- 0.5 ns, a mplitude = 0.76, and tau(2) = 6.0 +/- 0.2 ns, amplitude = 0.24). Very similar double-exponential fluorescence decays have been obtained with bound MANT-dADP and MANT-dATP. The data indicate that the chromophore s attached to the ribose experience two different environments when bo und to the DnaB helicase which may reflect the existence of two differ ent conformations of the ribose-binding region of the DnaB helicase nu cleotide-binding site. This result is in contrast to a single environm ent and probably a lack of discrete conformational heterogeneity at th e base-binding region, as probed by epsilon ADP fluorescence. Thus, th e data indicate that conformational heterogeneity is localized around the ribose and not transmitted to the base-binding region, suggesting limited communication between base- and ribose-binding sites. The repo rted results provide the first insight into the nature of the signific ant structural differences between base- and ribose-binding regions of the nucleotide-binding site of the DnaB helicase. The importance of t hese differences, in terms of the biological functioning of the DnaB p rotein, is discussed.