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Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei.

Amaro RE, Swift RV, McCammon JA - PLoS Negl Trop Dis (2007)

Bottom Line: Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite.Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists.Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA. ramaro@mccammon.ucsd.edu

ABSTRACT
RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

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Related in: MedlinePlus

RNA editing ligase 1 reaction and structure.A) The three-step ligation reaction catalyzed by RNA editing ligase 1. B) The energy minimized TbREL1 structure is depicted with ATP-bound in the active site. The crystallographic water molecules and explicit solvent are included in the simulations, but omitted in the figure for clarity. The five conserved ligase motifs are colored and labeled, and key conserved residues within each motif are depicted in licorice.
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pntd-0000068-g001: RNA editing ligase 1 reaction and structure.A) The three-step ligation reaction catalyzed by RNA editing ligase 1. B) The energy minimized TbREL1 structure is depicted with ATP-bound in the active site. The crystallographic water molecules and explicit solvent are included in the simulations, but omitted in the figure for clarity. The five conserved ligase motifs are colored and labeled, and key conserved residues within each motif are depicted in licorice.

Mentions: The remarkable mRNA editing process begins in the trypanosomal mitochondrial (mt) DNA, which consists of a topologically linked network of thousands of minicircles and dozens of maxicircles. It is the transcripts of these maxicircles, encoding components of respiratory complexes and energy transduction systems, which undergo extensive RNA editing. The editing process begins when guide RNAs (gRNAs) are transcribed from the minicircles in the mt genome and subsequently base-pair with pre-mRNA sequences through a conserved “anchor sequence” [9],[10]. Endonucleolytic cleavage of the pre-mRNA strand occurs at a point of mismatch between the trans-acting gRNA and its cognate pre-mRNA, by endonucleolytic enzymes that have yet to be characterized. Depending on the type of RNA mismatch, U's are either added, by terminal uridylyl transferase (TUTase), or deleted, by a U-specific 3′ exonuclease (ExoUase). The processed RNA fragments are then religated by one of two RNA ligases, Kinetoplastid RNA editing ligase 1 (TbREL1) or RNA editing ligase 2 (TbREL2), generally depending on whether the process is deletion or insertion editing, respectively. Religation of the now completely base-paired double stranded RNA (dsRNA) strands occurs in a three-step process (Fig. 1A). In the first step, the catalytic lysine residue acts in concert with a divalent metal cofactor within the ATP binding pocket of TbREL1 and is autoadenylated, forming a protein-AMP intermediate and releasing pyrophosphate. In the next step, a 5′-5′ phosphate linkage is formed when the AMP is transferred to the 5′ end of the nicked 3′ RNA substrate, which is proximal to the active site. Finally, the ligation process is completed when the 3′ hydroxyl group of the other strand displaces the 5′ AMP, resulting in a new phosphodiester bond.


Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei.

Amaro RE, Swift RV, McCammon JA - PLoS Negl Trop Dis (2007)

RNA editing ligase 1 reaction and structure.A) The three-step ligation reaction catalyzed by RNA editing ligase 1. B) The energy minimized TbREL1 structure is depicted with ATP-bound in the active site. The crystallographic water molecules and explicit solvent are included in the simulations, but omitted in the figure for clarity. The five conserved ligase motifs are colored and labeled, and key conserved residues within each motif are depicted in licorice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2100368&req=5

pntd-0000068-g001: RNA editing ligase 1 reaction and structure.A) The three-step ligation reaction catalyzed by RNA editing ligase 1. B) The energy minimized TbREL1 structure is depicted with ATP-bound in the active site. The crystallographic water molecules and explicit solvent are included in the simulations, but omitted in the figure for clarity. The five conserved ligase motifs are colored and labeled, and key conserved residues within each motif are depicted in licorice.
Mentions: The remarkable mRNA editing process begins in the trypanosomal mitochondrial (mt) DNA, which consists of a topologically linked network of thousands of minicircles and dozens of maxicircles. It is the transcripts of these maxicircles, encoding components of respiratory complexes and energy transduction systems, which undergo extensive RNA editing. The editing process begins when guide RNAs (gRNAs) are transcribed from the minicircles in the mt genome and subsequently base-pair with pre-mRNA sequences through a conserved “anchor sequence” [9],[10]. Endonucleolytic cleavage of the pre-mRNA strand occurs at a point of mismatch between the trans-acting gRNA and its cognate pre-mRNA, by endonucleolytic enzymes that have yet to be characterized. Depending on the type of RNA mismatch, U's are either added, by terminal uridylyl transferase (TUTase), or deleted, by a U-specific 3′ exonuclease (ExoUase). The processed RNA fragments are then religated by one of two RNA ligases, Kinetoplastid RNA editing ligase 1 (TbREL1) or RNA editing ligase 2 (TbREL2), generally depending on whether the process is deletion or insertion editing, respectively. Religation of the now completely base-paired double stranded RNA (dsRNA) strands occurs in a three-step process (Fig. 1A). In the first step, the catalytic lysine residue acts in concert with a divalent metal cofactor within the ATP binding pocket of TbREL1 and is autoadenylated, forming a protein-AMP intermediate and releasing pyrophosphate. In the next step, a 5′-5′ phosphate linkage is formed when the AMP is transferred to the 5′ end of the nicked 3′ RNA substrate, which is proximal to the active site. Finally, the ligation process is completed when the 3′ hydroxyl group of the other strand displaces the 5′ AMP, resulting in a new phosphodiester bond.

Bottom Line: Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite.Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists.Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, USA. ramaro@mccammon.ucsd.edu

ABSTRACT
RNA editing ligase 1 (TbREL1) is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD) simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

Show MeSH
Related in: MedlinePlus