The existence and widespread occurrence of several devastating trypanosomal tropical diseases, such as Chagas' disease and African sleeping sickness, cause an estimated 1 million deaths each year in developing countries 
. In 2005, the completely sequenced genomes of Trypanosoma brucei
, the causative agent of African sleeping sickness, T. cruzi
, the causative agent of Chagas disease, and Leishmania major
, the causative agent of Leishmaniasis, were published, yet, despite these great genomic successes, the need for effective and suitable drugs still remains 
. Currently available drugs were developed in the first half of the twentieth century and they are toxic, difficult to deliver and often ineffective 
The trypanosome pathogens responsible for these diseases all share unique post-transcriptional mRNA editing features, the discovery of which revealed a rich addition to the central dogma of biology, in which information not only passes from DNA to RNA to protein, but also between different classes of RNA 
. Through the insertion and deletion of uridylates (U's), the editing process transforms premature mitochondrial RNA (pre-mRNA) to mature mRNA in a multi-protein complex known as the editosome 
. The exact composition of the editosome complex has yet to be fully characterized, although 20S core complexes have a Mw
of 1.6 MDa and appear to be comprised of 16-20 proteins 
. To further complicate matters, it has recently been demonstrated that at least three different 20S editosomes of heterogeneous composition and distinct specificity are involved in the editing process 
, possibly reflecting compositional changes of this dynamic multicatalyst complex at different stages in the editing process 
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” 
. 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 (). 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.
RNA editing ligase 1 reaction and structure.
TbREL1 has been shown to play a key role in the vitality of Trypanosoma brucei
, as it is required for survival of both the insect and bloodstream forms of the pathogen 
. TbREL1 is comprised of a catalytic N-terminal adenylation domain and a C-terminal domain that facilitates non-covalent interaction with another editosome protein, KREPA2 
. Interestingly, an oligonucleotide binding (OB-fold) domain, usually associated with DNA ligases and capping enzymes in cis
, appears to be provided by KREPA2 in trans
and has been predicted to act as a conformational switch regulating various steps in the editing process 
. The recently crystallized 1.2 Å resolution structure of the adenylation domain, TbREL1, from T. brucei
with the bound ATP ligand revealed a number of interesting features of the enzyme, including an unusual water-solvated ATP binding pocket 
. As the N-terminal domain alone has shown to be capable of RNA ligation activity, further studies characterizing its functional dynamics are relevant. Interestingly, although a single Mg2+
ion is clearly coordinated to the protein and ATP in the crystal structure, the protein is not yet adenylated, indicating that the structure is in a pre-catalytic conformation. Furthermore, it has been suggested for this system, and shown experimentally for related systems, that the nucleotidyl transfer reaction proceeds by a two Mg2+
. It has been hypothesized that the first Mg2+
binds with high affinity between the ATP β− and γ−phosphate groups, as seen in the TbREL1 crystal structure, and the second magnesium coordinates to a lower-affinity site between the ATP α-phosphate group and catalytic lysine, subsequently promoting the bond cleavage between the α and β phosphate and the adenylyl transfer 
TbREL1 belongs to the covalent nucleotidyl transferase superfamily of enzymes, along with other RNA ligases, mRNA capping enzymes, and DNA ligases 
. A thorough bioinformatics and phylogenetic analysis of the RNA ligase family shows five well-conserved structural motifs responsible for the three-step nucleic acid repair and strand-joining reaction, a shared overall protein fold, and common evolutionary traces. At the level of the superfamily, the percent identity among the sequences is less than 10%, which renders traditional sequence alignment measures ineffective. However, a structural alignment of an unbiased and nonredundant set of the members of the superfamily, which includes RNA ligase II, mRNA capping enzyme, NAD+ dependent DNA ligase and the ATP dependent DNA ligase, indicates that there is a well-conserved core structure surrounding the nucleotide binding site (). Furthermore, this analysis reveals eight highly conserved residues that may play key catalytic roles. The structural differences within the nucleotide binding sites are likely due to the subtle differences in their cofactor specificities. A structural phylogenetic analysis based on the multiple structural alignment of the superfamily indicates that the closest known relative to TbREL1 is T4 phage RNA ligase 2 (Fig. S1
), and this is consistent with a previous sequence-based evolutionary analysis of the family 
. Steady-state and pre-steady-state kinetic analysis coupled with strategic mutagenesis of the T4 RNA ligase 2 has established functional roles for the highly conserved binding site residues and mapped many of the important interactions of the RNA ligase active site 
. The established similarity between these two enzymes is important, as much of this information for the T4 phage system can be used to help interpret, understand, and direct strategic studies for the enzymatic activity of TbREL1.
Structural alignment of the covalent nucleotidyl transferase superfamily.
The existence of a high-resolution ligand-bound crystal structure and unique active site features, coupled with the fact that there is no close human homolog, make TbREL1 an important target for development of inhibitors against these protozoan parasites. In this work, we use all-atom explicit solvent molecular dynamics (MD) simulations to probe the structure, function, and dynamics of TbREL1 on the nanosecond timescale. A comprehensive structural and sequence alignment of all the known superfamily members identifies several key residues that we monitor throughout the simulations. Deeply buried water molecules within the nucleotide binding site and their effect on the mode of ATP-binding are also investigated. A comparison of the principal components for the apo and ATP-bound systems illustrates large local and global differences in the enzyme motion. Ensemble-averaged electrostatics calculations from the MD simulations reveal a putative RNA binding site near the ligase active site, a finding that has previously eluded scientists. Coupled with the crystal structure and currently available experimental information, the dynamics and structural analysis presented here will likely prove to be salient aspects of future successful drug design efforts against this vital enzyme.