X-ray crystallographic studies of HhaI DNA (cytosine-C5)-methyltransferase (M.HhaI) covalently linked to methylated 5-fluorocytosine in DNA provided the first direct evidence that the cytosine residue targeted for methylation was "flipped" out of the helix during the transfer reaction. Subsequent studies indicated that removal of the target cytosine base, i.e., introduction of an abasic site, enhanced binding of M.HhaI to DNA and that the conformation of the sugar-phosphate backbone at the abasic site in the resultant complexes was the same as that of the sugar attached to a "flipped" cytosine. In the present study, pseudorotationally constrained sugar analogues, based on bicyclo[3.1.0]hexane templates, were placed in DNA duplexes as abasic target sites in the M.HhaI recognition sequence. Biochemical studies demonstrate that binding affinity of M.HhaI for abasic sites increases when the abasic target sugar analogue is constrained to the south conformation and decreases when it is constrained to the north conformation. In native gel-shift assays, M.HhaI exhibits a "closed" conformation when bound to the abasic south or abasic furanose analogues, whereas an "open" conformation predominates with the abasic north analogue. A structural understanding of these results was obtained via molecular dynamics simulations of the DNA duplex alone and in ternary complex with M.HhaI and cofactor, along with quantum mechanical calculations on model compounds representative of the abasic and modified sugars. Binding affinities are shown to be related to the ability of the abasic sugar analogues to spontaneously flip out of the DNA duplex. Enhanced binding of the abasic south analogue is suggested to be due to its increased capacity for sampling the experimentally observed conformation of the DNA target site in the M.HhaI ternary complex. Decreased binding of the north analogue is due to decreased flexibility of the phosphodiester backbone associated with a north pseudorotation angle, thereby inhibiting spontaneous flipping of the sugar moiety out of the DNA duplex. Spontaneous flipping of the sugar moiety out of the DNA duplex is also suggested to facilitate formation of a "closed" complex between M.HhaI and DNA whereas partial or no flipping favors the "open" conformation. These results show that introduction of structural constraints into DNA that induce enhanced sampling of protein-bound conformations facilitate DNA-protein binding. Implications of the present results with respect to the mechanism of base flipping in the M.HhaI catalytic cycle are discussed.