Fig 1.
Size exclusion chromatography (SEC) analyses of RESC5 reveals monomer.
A SEC analysis of the T. brucei RESC5. The SEC profile is shown. B SEC analyses of MW. The x and y axes are Log MW and column volume, respectively. RESC5 eluted at calculated MWs of 30 kDa (yellow triangle). The standards used for calculation of the standard curve are shown as blue diamonds and were aproptinin (6.5 kDa), cytochrome c oxidase (12.4 kDa), carbonic anhydrase (29 kDa), and albumin (66 kDa).
Fig 2.
Crystal structure of T. brucei RESC5.
Cartoon figure of RESC5 with strands colored magenta and helices, cyan. Shown are two views of the structure related by a 90° rotation about the y axis. Secondary structure elements are labeled in the view on the left. All ribbon diagrams were made using PyMOL [58].
Table 1.
Data collection and refinement statistics: T. brucei RESC5.
Fig 3.
T. brucei RESC5 has a DDAH fold.
A Overlay of T. brucei RESC5 (pink) and bovine DDAH (pdb code: 2CI6) structures (green). The overlay shows that although the positioning of secondary structural show some differences, they both contain the same overall fold. B Close up of the overlay from Fig 3A showing positions of key active site residues in DDAH. Also shown as sticks and labeled is the citrulline bound in the DDAH structure. While important arginine and aspartic acid residues in the active sites are conserved between the two proteins, central catalytic residues from the catalytic triad Cysteine-Histidine are Alanine-Proline in RESC5. C Structure based sequence alignment of T. brucei RESC5 with DDAH. DDAH used is pdb id code: 2CI6 (from the alignment shown in Fig 3A). In the sequence alignment conserved residues between the two proteins are colored grey. Yellow residues are those involved in catalysis or substrate/product binding by DDAH. Note, several of these residues are conserved in RESC5. However, notably the catalytic cysteine and histidine (asterisks) are not.
Fig 4.
Thermal shift assays analyzing RESC5 binding to DDAH substrate and product.
A Melting curves for 10 μM WT RESC5 in the presence of varying concentrations of L-citrulline and dimethylaminoarginine. Data was scaled from 0–1. B Melting curves for 10 μM RESC5(R71A-P180H-S277C) in the presence of varying concentrations of L-citrulline and dimethylaminoarginine. Data was scaled from 0–1. Error bars represent standard deviations from the average (of at least three technical triplicate runs).
Fig 5.
Multiple sequence alignment of putative RESC5 homologs and conserved surface residues.
Homolog IDs are indicated to the left. Secondary structural information from the crystal structure is shown above sequence blocks and key residues that interact with RESC6 are boxed and the interface they mediate contacts with (1 or 2) are indicated under the alignment. The proteins are: XP_823393.1; hypothetical protein, conserved [Trypanosoma brucei brucei TREU927; RESC5], the protein under study, RHW69169.1; Mitochondrial RNA binding protein [Trypanosoma brucei equiperdum]. KAG8344931.1; putative amidinotransferase [Trypanosoma vivax], KAH9578299.1; hypothetical protein LSM04_001006 [Trypanosoma melophagium], XP_028884646.1 putative amidinotransferase [Trypanosoma theileri], XP_029237384.1; putative amidinotransferase [Trypanosoma rangeli], XP_811845.1; hypothetical protein [Trypanosoma cruzi strain CL Brener], KAG5495086.1; hypothetical protein JKF63_02139 [Porcisia hertigi], XP_010702224.1; mitochondrial RNA binding protein, putative [Leishmania panamensis], XP_015655064.1 putative mitochondrial RNA binding protein [Leptomonas pyrrhocoris]. B surface representation of the RESC5 structure colored according to conservation (from the alignment in Fig 5A). Pink regions represent highly conserved regions while dark grey are not conserved. Shown are two “sides” of the molecule. The pocket corresponding to the active site pocket in DDAH is labeled “pocket” and is conserved.
Fig 6.
Electrostatic surface potential of RESC5 reveals potential nucleic acid binding patches.
A RESC5 displayed as a ribbon diagram with the residues corresponding to the catalytic cysteine in DDAH enzymes (an alanine in RESC5) colored red. B RESC5 shown as an electrostatic surface representation with blue and red regions representing positive and negative regions, respectively.
Fig 7.
Phylogenetic trees for RESC5 and DDAH.
A Phylogenetic tree for the RESC5 proteins. The species names are indicated in each branch and the timescale (1000 years) for the branches are included. B Phylogenetic tree for DDAH. DDAH proteins were only identified in Chordata (magenta), Eubacteria (green) and Arthropoda (blue). Representative species in each phyla are indicated.