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Potential regulatory interactions of Escherichia coli RraA protein with DEAD-box helicases.

Pietras Z, Hardwick SW, Swiezewski S, Luisi BF - J. Biol. Chem. (2013)

Bottom Line: We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB.Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å.The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, United Kingdom and.

ABSTRACT
Members of the DEAD-box family of RNA helicases contribute to virtually every aspect of RNA metabolism, in organisms from all domains of life. Many of these helicases are constituents of multicomponent assemblies, and their interactions with partner proteins within the complexes underpin their activities and biological function. In Escherichia coli the DEAD-box helicase RhlB is a component of the multienzyme RNA degradosome assembly, and its interaction with the core ribonuclease RNase E boosts the ATP-dependent activity of the helicase. Earlier studies have identified the regulator of ribonuclease activity A (RraA) as a potential interaction partner of both RNase E and RhlB. We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB. Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å. The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases.

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Related in: MedlinePlus

Crystal structure of RraA in complex with the C-terminal extension of RhlB (RhlB(398–421)).A, the RhlB fragment is shown in sticks representation (green). The black mesh represents a 2Fo − Fc electron density map. RraA is colored red. B, superposition of SrmB CTD (blue) bound to RraA on the RhlB fragment. The RraA trimer is shown in red, and RhlB(398–421) is shown in green.
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Figure 4: Crystal structure of RraA in complex with the C-terminal extension of RhlB (RhlB(398–421)).A, the RhlB fragment is shown in sticks representation (green). The black mesh represents a 2Fo − Fc electron density map. RraA is colored red. B, superposition of SrmB CTD (blue) bound to RraA on the RhlB fragment. The RraA trimer is shown in red, and RhlB(398–421) is shown in green.

Mentions: The electron density maps were clear and well resolved for all the RraA protomers. Poorer quality electron density was identified on the surface of a single RraA protomer that is able to accommodate a single RhlB(398–421) peptide (Fig. 4A). The identified density is likely to originate from RhlB(398–421) as there are no elongated molecules (e.g. polyethylene glycol) in the crystallization condition that may account for the continuous density. Due to the poor quality of the density it was not possible to model amino acid side chains for the peptide, but 15 of 23 main chain residues could be traced with confidence (two chains of 6 and 9 residues, Fig. 4A). The two segments of the traced main chain likely belong to one molecule; however, the discontinuity in the electron density did not allow the fragments to be joined with confidence. The density is located close to electronegative patches on the RraA surface, which is compatible with the complementary electropositive nature of RhlB(398–421) (predicted pI 12.5). As the peptide side chains could not be modeled unequivocally, it is not possible to derive detailed information about the RhlB(398–421)/RraA interface. Nonetheless, the structure indicates an approximate site of RhlB fragment binding on the surface of RraA. Surprisingly, the contact site overlaps with the interface identified for the SrmB/RraA assembly (Fig. 4B) and the modeled RhlB residues lay in proximity of RraA residues Asp-50 and Asp-128. This suggests that RraA harbors one surface region that is the binding site for both RhlB and SrmB.


Potential regulatory interactions of Escherichia coli RraA protein with DEAD-box helicases.

Pietras Z, Hardwick SW, Swiezewski S, Luisi BF - J. Biol. Chem. (2013)

Crystal structure of RraA in complex with the C-terminal extension of RhlB (RhlB(398–421)).A, the RhlB fragment is shown in sticks representation (green). The black mesh represents a 2Fo − Fc electron density map. RraA is colored red. B, superposition of SrmB CTD (blue) bound to RraA on the RhlB fragment. The RraA trimer is shown in red, and RhlB(398–421) is shown in green.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814787&req=5

Figure 4: Crystal structure of RraA in complex with the C-terminal extension of RhlB (RhlB(398–421)).A, the RhlB fragment is shown in sticks representation (green). The black mesh represents a 2Fo − Fc electron density map. RraA is colored red. B, superposition of SrmB CTD (blue) bound to RraA on the RhlB fragment. The RraA trimer is shown in red, and RhlB(398–421) is shown in green.
Mentions: The electron density maps were clear and well resolved for all the RraA protomers. Poorer quality electron density was identified on the surface of a single RraA protomer that is able to accommodate a single RhlB(398–421) peptide (Fig. 4A). The identified density is likely to originate from RhlB(398–421) as there are no elongated molecules (e.g. polyethylene glycol) in the crystallization condition that may account for the continuous density. Due to the poor quality of the density it was not possible to model amino acid side chains for the peptide, but 15 of 23 main chain residues could be traced with confidence (two chains of 6 and 9 residues, Fig. 4A). The two segments of the traced main chain likely belong to one molecule; however, the discontinuity in the electron density did not allow the fragments to be joined with confidence. The density is located close to electronegative patches on the RraA surface, which is compatible with the complementary electropositive nature of RhlB(398–421) (predicted pI 12.5). As the peptide side chains could not be modeled unequivocally, it is not possible to derive detailed information about the RhlB(398–421)/RraA interface. Nonetheless, the structure indicates an approximate site of RhlB fragment binding on the surface of RraA. Surprisingly, the contact site overlaps with the interface identified for the SrmB/RraA assembly (Fig. 4B) and the modeled RhlB residues lay in proximity of RraA residues Asp-50 and Asp-128. This suggests that RraA harbors one surface region that is the binding site for both RhlB and SrmB.

Bottom Line: We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB.Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å.The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, United Kingdom and.

ABSTRACT
Members of the DEAD-box family of RNA helicases contribute to virtually every aspect of RNA metabolism, in organisms from all domains of life. Many of these helicases are constituents of multicomponent assemblies, and their interactions with partner proteins within the complexes underpin their activities and biological function. In Escherichia coli the DEAD-box helicase RhlB is a component of the multienzyme RNA degradosome assembly, and its interaction with the core ribonuclease RNase E boosts the ATP-dependent activity of the helicase. Earlier studies have identified the regulator of ribonuclease activity A (RraA) as a potential interaction partner of both RNase E and RhlB. We present structural and biochemical evidence showing how RraA can bind to, and modulate the activity of RhlB and another E. coli DEAD-box enzyme, SrmB. Crystallographic structures are presented of RraA in complex with a portion of the natively unstructured C-terminal tail of RhlB at 2.8-Å resolution, and in complex with the C-terminal RecA-like domain of SrmB at 2.9 Å. The models suggest two distinct mechanisms by which RraA might modulate the activity of these and potentially other helicases.

Show MeSH
Related in: MedlinePlus