Structural characterization of transient reaction intermediates is a formidable challenge, made even more difficult in protein-DNA interactions by the occurrence of weak enzyme binding modes that thwart formation of the unique complex of interest. Here we report the first use of a reaction coordinate tuning method to populate a transitory state during enzymatic base flipping (). The approach is based on two principles obtained from extensive thermodynamic, NMR and rapid kinetic studies of base flipping by UNG 8–11
. The first principle is that the equilibrium constant for base flipping may be shifted away from the reactant state by destabilization of the uracil base pair. Accordingly, the nonpolar adenine isostere 4-methylindole (M) possesses no hydrogen bonding groups () 12
, and when placed opposite to uracil in DNA, allows UNG to bind 8,000 times more tightly as compared to an identical DNA with a U:A base pair 10
. The second principle is that the fully extrahelical product state (FF, ) can be destabilized by replacing the 5-hydrogen of uracil with a bulky methyl group to make 5-methyluracil (5-MeU or T) that sterically prevents access to the uracil pocket () 7
. Combining these two principles flattens the energy landscape for base flipping and allows population of any transient extrahelical states that may exist between the reactant and product states (EI′, ).
The structure of human UNG bound to DNA containing a central T:M base pair refined to 2.45 Å resolution reveals that the central thymine is rotated from the base stack by about 30° (, Supplementary Fig. 2A
), which is only one-sixth of the 180° rotation required to fully flip uracil into the active site pocket (pdb code 1EMH, )6
. Hence this structure represents a structural snapshot of a very early intermediate on the base flipping pathway. Despite the large differences in the positions of the extrahelical bases in the early intermediate (EI′) and the previously reported fully flipped (FF) structure, the complexes share many of the same DNA backbone interactions, indicating that the initial steps in flipping uracil and thymine are identical (). Most notably, a key DNA intercalating residue (Leu272) protrudes into the minor groove of the DNA in both complexes (shown in yellow in and ), with its δCH3
group lodged within van der Waals contact distance of the deoxyribose ring of the flipped nucleotide. This leucine has been shown in rapid kinetic studies to be important in promoting forward migration along the base flipping pathway, with functional roles both early and late in the process 13
. The intercalated position of Leu272 in the EI′ complex is fully consistent with its proposed early role in flipping that involves plugging the gap in the base stack left behind by the extruded base, thereby inhibiting its retrograde motion back into the duplex. Additional interactions common to the EI’ and FF complexes involve the phosphodiester groups flanking the extrahelical nucleotide as shown in for the EI’ complex. These interactions include neutral hydrogen bonds from either the backbone amide or side chain hydroxyl groups of residues Ser169, Ser270, Ser273 and Ser247 to the bridging or nonbridging phosphate oxygens (Supplementary Fig. 2A
). The early emergence of these serine-phosphate interactions along the flipping pathway is consistent with previous kinetic and mutational studies13
Stabilization of extrahelical thymine in the EI′ state in complex with UNG
In summary, we conclude that T and U follow the same flipping reaction coordinate based on the following lines of evidence. First, both bases emerge spontaneously from the duplex with similar rates kinetically competent for base flipping (see further discussion below) 8, 9
. Second, the enzyme-DNA backbone interactions of T in the EI state are shared with that of U in the final FF state (see above). It is thus quite easy to envision how these early interactions would lead to the FF state. Third, there are no interactions of the enzyme with the substituent at the 5 position of the base in the EI state. Thus, there is no obvious way that UNG could discriminate between the 5-CH3
of T and the 5-H U at this stage, strongly suggesing that U and T occupy this same transient site (). Finally, we have also obtained a crystal structure of the abasic product DNA arising from slow excision of thymine in the crystal over several weeks (Supplementary Figure 2B
). Thus, T is a very slow substrate which requires that it transiently accesses the active site. Therefore, the pathway for flipping T is productive, consistent with it following the same pathway as U.
Despite similar DNA backbone interactions in both complexes, the overall DNA structures are quite different for the EI′ and FF complexes (, ). For both structures, the DNA resembles B-form DNA 3′ of the flipped nucleotide, with an average rise of ~ 10.5 base pairs per turn and standard 2′ endo sugar puckers. However, there is a ~ 20 ° shift in the helical axis immediately 5’ to the flipped uracil in the FF complex which arises from the extreme conformation of the extrahelical nucleotide. In contrast, the EI′ complex deviates in only a minor way from docked B-form DNA except for local perturbations at the immediate site of thymine extrusion (compare and ). The absence of extreme DNA bending in the EI′ complex is consistent with fluorescence and NMR studies of the early stages of base flipping that indicated little perturbation of the DNA base stack, and preservation of the B-form DNA conformation 8
. This aspect of the UNG reaction differs from observations obtained from structural studies of DNA complexes with human 8-oxoguanine DNA glycosylase (hOGG1) where the enzyme appears to bend DNA regardless of whether the cognate oxidized base (8-oxoguanine) is present 14, 15
. DNA bending is a key component of the base extrusion process because it allows widening of the major groove, providing an egress pathway for the base, and also serves to release some of the strain resulting from the DNA backbone distortions that accompany flipping.
The most striking difference between the EI′ and FF complexes is the position of the extrahelical thymine and uracil base (). The thymine base, which is highly exposed to solvent, docks against two regions of UNG that form the mouth of the active site. A key interaction is a charged hydrogen bond (d
= 2.6 Å) that is formed between thymine O2 and the side chain NHε
proton of the completely conserved His148, which is located near the beginning of a long coiled region of the protein backbone that stretches from residues Gln144 to Pro167 (red strand, ). A second interaction is observed between the imino proton of thymine and the backbone carbonyl of His212 (d
= 3.2 Å), which is located in a highly conserved nine residue turn that encompasses residues Ala211 to Glu219. Aside from these two hydrogen bonds, there are no other interactions of UNG with the thymine base. Nevertheless, these limited interactions could provide specificity for uracil and thymine because neutral cytosine is not complementary with this hydrogen bond donor-acceptor pattern. In addition, bulky purines would be sterically excluded from the site due to the tight packing of Leu272 against the deoxyribose, which fixes the position of the deoxyribose-base glycosidic bond. In contrast with the relatively sparse interactions observed in EI′, the uracil base is extensively desolvated in the FF complex, with every potential hydrogen bond donor and acceptor interaction fulfilled, and in addition, favorable edge-face aromatic interaction between Phe158 and the uracil ring 6
. The increasing interactions with the base and the phosphate backbone as the reaction proceeds indicates that base flipping occurs within an enzyme energy landscape that serves to drive the reaction forward in a succession of energetically downhill steps 10, 13