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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

Comparison of the RraA/SrmB structure and other DEAD-box protein complexes.A, B RraA bound to SrmB would prevent RecA-like domain closure due to steric clash (highlighted by dashed frame). SrmB is colored blue, the RraA protomer interacting with SrmB is shown in red and the other RraA protomers are shown in gray. The Dbp5 structure in closed conformation (PDB code 3FHT), superimposed onto SrmB CTD is shown in cyan with RNA colored yellow and shown in stick representation. The root mean square deviation for the fit is 1.34 Å. B, RraA binding may impede RNA binding by SrmB. The RraA loop consisting of residues 53–59 would occlude the predicted RNA binding surface of SrmB (highlighted by dashed frame). C, comparison of the RraA-SrmB and Dbp5-NUP214 complexes. The reference frame for the overlay is the SrmB CTD (blue) and the Dbp5 NTD (cyan). RraA is red and NUP214 is yellow (PDB code 3FHC). D, comparison of the RraA-SrmB and eIF4A-PDCD4 complexes. Superposition of SrmB CTD (blue) on eIF4A NTD (cyan). PDCD4 is shown in yellow (PDB code 2ZU6). The overlays are shown from two viewpoints related by a 90° rotation.
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Figure 6: Comparison of the RraA/SrmB structure and other DEAD-box protein complexes.A, B RraA bound to SrmB would prevent RecA-like domain closure due to steric clash (highlighted by dashed frame). SrmB is colored blue, the RraA protomer interacting with SrmB is shown in red and the other RraA protomers are shown in gray. The Dbp5 structure in closed conformation (PDB code 3FHT), superimposed onto SrmB CTD is shown in cyan with RNA colored yellow and shown in stick representation. The root mean square deviation for the fit is 1.34 Å. B, RraA binding may impede RNA binding by SrmB. The RraA loop consisting of residues 53–59 would occlude the predicted RNA binding surface of SrmB (highlighted by dashed frame). C, comparison of the RraA-SrmB and Dbp5-NUP214 complexes. The reference frame for the overlay is the SrmB CTD (blue) and the Dbp5 NTD (cyan). RraA is red and NUP214 is yellow (PDB code 3FHC). D, comparison of the RraA-SrmB and eIF4A-PDCD4 complexes. Superposition of SrmB CTD (blue) on eIF4A NTD (cyan). PDCD4 is shown in yellow (PDB code 2ZU6). The overlays are shown from two viewpoints related by a 90° rotation.

Mentions: Based on our structural data, we propose a mechanism to explain how RraA impedes SrmB activity. Superposition of the SrmB-RraA complex structure onto the crystal structure of the human DEAD-box helicase Dbp5 in complex with RNA and an ATP analog (PDB code 3FHT) reveals that bound RraA would prevent SrmB from adopting the closed conformation seen for the Dbp5 structure (Fig. 6A). It is well known that a closed helicase conformation is required for ATP turnover and RNA unwinding (44). Closer inspection of the superimposed helicase structures reveals that bound RraA partially occludes the RNA binding surface of SrmB. If RNA were to be bound to SrmB in a similar manner to the interaction seen for Dbp5, it would clash sterically with a loop on the surface of RraA consisting of residues 53–59 (Fig. 6B). Based on these structural overlays, we propose that RraA can influence SrmB activity by sterically inhibiting substrate binding and preventing the enzyme from adopting the closed conformation required for ATP hydrolysis.


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

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

Comparison of the RraA/SrmB structure and other DEAD-box protein complexes.A, B RraA bound to SrmB would prevent RecA-like domain closure due to steric clash (highlighted by dashed frame). SrmB is colored blue, the RraA protomer interacting with SrmB is shown in red and the other RraA protomers are shown in gray. The Dbp5 structure in closed conformation (PDB code 3FHT), superimposed onto SrmB CTD is shown in cyan with RNA colored yellow and shown in stick representation. The root mean square deviation for the fit is 1.34 Å. B, RraA binding may impede RNA binding by SrmB. The RraA loop consisting of residues 53–59 would occlude the predicted RNA binding surface of SrmB (highlighted by dashed frame). C, comparison of the RraA-SrmB and Dbp5-NUP214 complexes. The reference frame for the overlay is the SrmB CTD (blue) and the Dbp5 NTD (cyan). RraA is red and NUP214 is yellow (PDB code 3FHC). D, comparison of the RraA-SrmB and eIF4A-PDCD4 complexes. Superposition of SrmB CTD (blue) on eIF4A NTD (cyan). PDCD4 is shown in yellow (PDB code 2ZU6). The overlays are shown from two viewpoints related by a 90° rotation.
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Related In: Results  -  Collection

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

Figure 6: Comparison of the RraA/SrmB structure and other DEAD-box protein complexes.A, B RraA bound to SrmB would prevent RecA-like domain closure due to steric clash (highlighted by dashed frame). SrmB is colored blue, the RraA protomer interacting with SrmB is shown in red and the other RraA protomers are shown in gray. The Dbp5 structure in closed conformation (PDB code 3FHT), superimposed onto SrmB CTD is shown in cyan with RNA colored yellow and shown in stick representation. The root mean square deviation for the fit is 1.34 Å. B, RraA binding may impede RNA binding by SrmB. The RraA loop consisting of residues 53–59 would occlude the predicted RNA binding surface of SrmB (highlighted by dashed frame). C, comparison of the RraA-SrmB and Dbp5-NUP214 complexes. The reference frame for the overlay is the SrmB CTD (blue) and the Dbp5 NTD (cyan). RraA is red and NUP214 is yellow (PDB code 3FHC). D, comparison of the RraA-SrmB and eIF4A-PDCD4 complexes. Superposition of SrmB CTD (blue) on eIF4A NTD (cyan). PDCD4 is shown in yellow (PDB code 2ZU6). The overlays are shown from two viewpoints related by a 90° rotation.
Mentions: Based on our structural data, we propose a mechanism to explain how RraA impedes SrmB activity. Superposition of the SrmB-RraA complex structure onto the crystal structure of the human DEAD-box helicase Dbp5 in complex with RNA and an ATP analog (PDB code 3FHT) reveals that bound RraA would prevent SrmB from adopting the closed conformation seen for the Dbp5 structure (Fig. 6A). It is well known that a closed helicase conformation is required for ATP turnover and RNA unwinding (44). Closer inspection of the superimposed helicase structures reveals that bound RraA partially occludes the RNA binding surface of SrmB. If RNA were to be bound to SrmB in a similar manner to the interaction seen for Dbp5, it would clash sterically with a loop on the surface of RraA consisting of residues 53–59 (Fig. 6B). Based on these structural overlays, we propose that RraA can influence SrmB activity by sterically inhibiting substrate binding and preventing the enzyme from adopting the closed conformation required for ATP hydrolysis.

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