Allosteric Autoinhibition Pathway in Transcription Factor ERG: Dynamics Network and Mutant Experimental Evaluations

Allosteric autoinhibition exists in many transcription factors. The ERG proteins exhibit autoinhibition on DNA binding by the C-terminal and N-terminal inhibitory domains (CID and NID). However, the autoinhibition mechanism and allosteric pathway of ERG are unknown. In this study we intend to elucidate the residue-level allosteric mechanism and pathway via a combined approach of computational and experimental analyses. Specifically computational residue-level fluctuation correlation data was analyzed to reveal detailed dynamics signatures in the allosteric autoinhibition process. A hypothesis of “NID/CID binding induced allostery” is proposed to link similar structures and different protein functions, which is subsequently validated by perturbation and mutation analyses in both computation and experiment. Two possible allosteric autoinhibition pathways of L286-L382-A379-G377-I360-Y355-R353 and L286-L382-A379-G377-I360-Y355- A351-K347-R350 were identified computationally and were confirmed by the computational and experimental mutations. Specifically we identified two mutation sites on the allosteric inhibition pathways, L286P/Q383P (NID/CID binding site) and I360G (pathway junction), which completely restore the wild type DNA binding affinity. These results suggest that the putative protein structure–function relationship may be augmented with a general relationship of protein “structure/fluctuation–correlation/function” for more thorough analyses of protein functions

Specific activities of WT and point mutants of DndA measured using in vitro cysteine desulfurase activity assay.

<p>Assays were performed for five times, and the average values of specific activities along with standard deviations of the measurements were shown.</p

Crystal structure of DndA from <i>Streptomyces lividans</i>.

<p>(<b>A</b>) <b>Overall structure of the DndA dimer.</b> The structure is viewed perpendicular to the two-fold axis of the dimer. The two protomers are shown in magenta and green, respectively. Their bound PLP cofactors are presented as sticks, with carbon atoms yellow, nitrogen atoms blue, oxygen atoms red, and phosphorus atoms orange. (<b>B</b>) <b>Structure of a protomer of DndA.</b> α helices are shown in cyan, β sheets are shown in magenta, and loops are shown in pink. PLP and its covalently linked Lys200 of DndA, as well as the catalytic Cys327 (mutated to serine in our study), are shown in stick representation.</p

The binding site of PLP on DndA.

<p>(<b>A</b>) <b>PLP is located in a deep surface pocket on DndA.</b> The two protomers of DndA are shown in surface representation, with only one PLP shown in stick representation. The protomer of DndA harboring this PLP is colored in light grey, whereas the other protomer is colored in dark grey. Blue, red, yellow, and orange represent nitrogen, oxygen, carbon, and phosphorus atoms, respectively. (<b>B</b>) <b>The interaction interface between PLP and DndA.</b> DndA is shown in grey, with carbon atoms of its side chains and PLP shown in green. Blue, red, and orange represent nitrogen, oxygen, and phosphorus atoms, respectively. Hydrogen bonds are represented by magenta dashed lines. The orange circle indicates the presumable location of the carboxylate group of the L-cysteine substrate.</p

Structural comparison of DndA with related cysteine desulfurases/selenocysteine lyase.

<p>(<b>A</b>) Structural superimposition of DndA (red), IscS (green, PDB code 1P3W), NifS (cyan, PDB code 1ECX), CsdB (magenta, PDB code 1C0N), and SufS (blue, PDB code 1T3I). Their bound PLP's are shown as sticks. (<b>B</b>) In DndA, the active site Cys327 is located on a β strand, and its distance from PLP is ∼16 Å. In IscS (<b>C</b>) and NifS (<b>D</b>), the active site cysteines are located on relatively long loops, and are not visible in the crystal structure. Visible residues closest to the catalytic cysteines on the primary sequence are no less than 9 Å from PLP. In CsdB (<b>E</b>) and SufS (<b>F</b>), the active site cysteines are located on relatively short loops, and are ∼7 Å from PLP.</p