Rotation of a DNA or RNA nucleotide out of the double helix and into a protein 
pocket ('base flipping') is a mechanistic feature common to some DNA/RNA-binding 
proteins. Here, we report the structure of HhaI methyltransferase in complex 
with DNA containing a south-constrained abasic carbocyclic sugar at the target 
site in the presence of the methyl donor byproduct AdoHcy. Unexpectedly, the 
locked south pseudosugar appears to be trapped in the middle of the flipping 
pathway via the DNA major groove, held in place primarily through Van der Waals 
contacts with a set of invariant amino acids. Molecular dynamics simulations 
indicate that the structural stabilization observed with the south-constrained 
pseudosugar will not occur with a north-constrained pseudosugar, which explains 
its lowered binding affinity. Moreover, comparison of structural transitions of 
the sugar and phosphodiester backbone observed during computational studies of 
base flipping in the M.HhaI-DNA-AdoHcy ternary complex indicate that the 
south-constrained pseudosugar induces a conformation on the phosphodiester 
backbone that corresponds to that of a discrete intermediate of the 
base-flipping pathway. As previous crystal structures of M.HhaI ternary complex 
with DNA displayed the flipped sugar moiety in the antipodal north conformation, 
we suggest that conversion of the sugar pucker from south to north beyond the 
middle of the pathway is an essential part of the mechanism through which 
flipping must proceed to reach its final destination. We also discuss the 
possibility of the south-constrained pseudosugar mimicking a transition state in 
the phosphodiester and sugar moieties that occurs during DNA base flipping in 
the presence of M.HhaI.