Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity

DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease

“…One structural difference was identified, which would impact complexes with secondary sequences substituted in the first bp, and another that would impact those with the substitution in the second position (as used in this study). 34,36 This latter change occurs in the DNA and results in greater stacking of the second and third base pairs of the recognition sequence in the H conformation compared to the L (Figure 1E). 34 Because the identity of the second nucleotide differs in the two types of sequences (C in the secondary sequence and A or G in the primary sequence), the energy associated with the difference in stacking will also differ.…”

“…34−36 Hence, filamentation stabilizes this activated (H state) conformation of SgrAI, which positions a second divalent cation near the DNA leading to rapid DNA cleavage. 36 In addition to changes in the structure of SgrAI and the SgrAI-DNA interface, differences in the structure of the bound DNA between the L and H states were also observed. 34 One change that appears especially relevant for the differences in behavior with primary and secondary recognition sequences is a change in base stacking between the second and third nucleotides of the bound recognition sequence (Figure 1E).…”

“…Structures of SgrAI bound to DNA have been solved in filamentous forms (the high activity or H state, or in prior work, the R state), and in the nonfilamentous, dimeric state (the low activity or L state, or in prior work, the T state). 34,36,42,43 Comparison of the representative L and H states shows an 11°rotation of one chain of the dimer relative to the other along an axis roughly perpendicular to the bound DNA helical axis 42 (Figure 1C). To accommodate this intersubunit rotation, residues at the dimeric interface adopt new orientations, which propagate to the active site where DNA cleavage takes place.…”

“…34 These shifts create a new binding pocket for a second divalent cation immediately adjacent to the scissile phosphate (i.e., the moiety containing the phosphodiester bond to be cleaved) in each active site (Figure 1D). 34,36 The nonfilamentous structure (the L state, left panel, Figure 1D) shows binding of only a single divalent cation near the DNA; however, two are expected in the two-metal ion mechanism for DNA cleavage. 44−46 Therefore, the second divalent cation that binds only in the filamentous conformation (the H state, right panel, Figure 1D) is expected to be the origin of the accelerated DNA cleavage activity of SgrAI.…”

“…Why these enzymes form polymeric filaments and/or cellular self-assemblies remains an important question. Some hypotheses include rapid activation or inactivation of enzymatic activity, modulation of allosteric responses, storage and protection from autophagy, control of cellular localization, and/or buffering of enzyme activity in cells. , While the structures of these filamentous enzymes are now being revealed, − including for the SgrAI system, − far fewer studies have been performed to determine the underlying kinetic mechanisms of the phenomenon.…”