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Filament_DBD_smaller_file.mp4

Can an enzyme be activated, and have its substrate specificity changed by forming a filament?

Yes! We study the enzyme SgrAI, which forms filaments (see movie to the left). When in the filament, it cleaves DNA 200-1000 times faster, and it even cleaves additional DNA sequences not cleaved when it isn't filamented. How cool is that? People told me I was crazy for thinking this, but the data doesn't lie!

Below is a movie showing the differences in conformations of the DNA nuclease SgrAI when in the filamentous and nonfilamentous forms:

Conformational_changes_accompany_filamentation.mp4

Enzyme Filamentation by SgrAI

The Horton Lab studies filament forming enzymes in order to understand the purpose of enzyme filamentation and its effect on enzyme function.
These studies combine biochemical, biophysical, biological, and structural approaches.
One filament forming enzyme we study is SgrAI, a sequence specific DNA endonuclease that is activated when in the filamentous form.
Fascinatingly, its DNA sequence specificity also changes when filamented.
he SgrAI forms only under particular conditions to maximize cleavage of invading DNA, and minimize damage to its host.

Conformational_changes_lead_to_important_changes_in_active_site.mp4

Structural Mechanism of SgrAI Activation by Filamentation

To the left is a movie showing the structural changes in the active site of SgrAI upon filamentation. In the non-filamentous form, the second required Mg2+ is distant from the DNA. Filamentation induces a twisting of the two subunits of the dimer, requiring an adjustment of a segment at the dimeric interface which connects directly to residues in the active site. This segment shifts towards the DNA, stabilizing the needed Mg2+.

Low_activity_state_active_site.mp4

Non-Filamentous Structural State of SgrAI Bound to DNA

To the left is a movie showing structure of SgrAI bound to DNA containing its primary recognition sequence determined by x-ray crystallography. This structure represents the low activity, non-filamentous state. Two to three divalent cations (Mg2+ in vivo) bind per active site to participate in catalyzing DNA bond cleavage. DNA cleavage by SgrAI is slow in this structural state, and nearly undetectable for SgrAI bound to its secondary recognition sequences. The structure of SgrAI bound to secondary recognition sequences looks nearly indistinguishable from the structure of SgrAI bound to its primary recognition sequence, in this low activity state. To unravel the mystery behind how filamentation changes SgrAI specificity to include cleavage of secondary site sequences, the cryo-electron microscopy structure of SgrAI bound to such sites within the filament is currently being studied.

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