Figure 5. (A) Projection of normal mode directions on the protein reaction coordinate corresponding to relaxation of positive (in blue) and negative (in red) supercoils. Reaction coordinate generated by fixing the upstream DNA and rotating the downstream part (see also Table 1) (B) Same as panel A except that the reaction coordinate of the protein was generated in two other independent runs, applying torque on both upstream and downstream DNA. supercoiled DNA, substrate discrimination can take place before strand passage (14). It is of considerable interest to explore whether this strategy is used by type I enzymes. DISCUSSION Conclusions of computational studies As a result of applications of MD simulations and of normal mode analysis, we have determined that: (i) The relaxation mechanisms for positive and negative supercoils are not the same. Removal of positive supercoils requires opening of the lips by 10–14 A˚ , while removal of negative supercoils needs the hinge region (from Leu429 to Lys436) to stretch about 12 A˚ . (ii) The N-terminal part of the protein, a subject of ongoing debate, is likely to be important for the relaxation of nega- tively supercoiled DNA, and it may or may not be impor- tant for the relaxation of positively supercoiled DNA. (iii) As suggested by structural data, the linker domain and the nose-cone helices are found to control the rotations needed to relax DNA. In additional insight, simulations reveal that Nucleic Acids Research, 2005, Vol. 33, No. 20 6631 the linker domain dominantly controls rotations for removal of positive supercoils, whereas the nose cones (particularly the left nose-cone helix) is the dominant con- troller of the rotations that remove negative supercoils. (iv) The protein has large, and almost identical, inherent struc- tural flexibility (as characterized by normal modes) towards both type of relaxations. On the kinetic effects of clamping Kinetic data are available for the related topo IB enzyme, vaccinia topoisomerase. For it, biochemical evidence shows that, on average, ﬁve strand rotations per binding event occur (68) [in contrast to a single-molecule estimate of about 19 rota- tions per cycle (24)], and that cleavage is slower than rotation and religation (32,69,70). However, no kinetic data are avail- able to discern what the rate limiting step is for the human enzyme under processive conditions (i.e. when it relaxes sev- eral supercoils upon one binding event) and physiological DNA substrate concentrations. Equally frustrating is also that no data exist to bear on the question of whether or not rotation is slower in either the distal or proximal clamp states. It is certainly possible that in the closed clamp conformation, rotation is impeded relative to the unclamped state. What will be important to consider experimentally is whether rotation is faster than religation since, once cleavage occurs, these are the two competing reactions. These parameters have not been measured for the human enzyme and are certainly more dif- ﬁcult to measure than for the vaccinia topo (J. J. Champoux, private communication). Moreover, it is not clear if conclu- sions from vaccinia topo I can be extrapolated to human topo I, because the former has pronounced sequence speciﬁcity and, presumably, a distinct covalent DNA-binding pattern invol- ving an extrahelical nucleotide position at the binding site (71). What will be important to address, in future computa- tional studies of the Cys-clamped complex, is to which extent rotation is impeded by the distal or the proximal strand. The possibility that a difference between the seemingly contradict- ory results obtained when disulphide-clamping the lips by Carey et al. (45) and by Woo et al. (46) is due to the absence and, respectively, the presence of the N-terminus in the two constructs cannot formally be excluded. However, because of the distinct positioning of the two clamps, we expect that the clamped simulations will reveal signiﬁcant dynamic and ener- getic differences of DNA interactions, in particular with the nose cone helices. Simulated mechanisms suggest additional experiments Our computational ﬁndings of the different mechanisms men- tioned above can be used to engineer topoisomerases with inhibited activity towards, or that selectively relax only, one kind of supercoils. One way this can be achieved is by engineering the proximal clamp and therefore preventing relaxation of positive supercoils. As we show that the lips do not open signiﬁcantly during relaxation of negative super- coils, this clamping should still permit relaxation of negative supercoils. In this case, one may observe a decrease in the rotation rate, as our MD simulations show that the distances corresponding to both the distal and the proximal clamps increase by 2 A˚ , and then decrease back to original values.