Crystallographic analysis of MjDim1
Protein expression and purification were carried out as previously described,5
except for the protein used to grow form 1 crystals of MjDim1. Form 1 crystals were grown from protein altered with mutations (K137A, E138A) thought to improve the chances and quality of crystallization by reducing surface entropy effects17
). Mutations were introduced using the Quick Change protocol (Stratagene) and the primers 5′- GCC AAG AGA ATG GTA GCT GCC GCG GGA ACA AAA GAT TAT GGA AGG -3′ and 5′- CCT TCC ATA ATC TTT TGT TCC CGC GGC AGC TAC CAT TCT CTT GGC -3′. Expressed protein of both forms was dialyzed into 50 mM Tris-HCl (pH 7.4), 50 mM NaCl and 5 mM 2-mercaptoethanol, and was concentrated to a value of 25-30 mg/ml prior to crystallization. Protein purity was assessed by SDS-PAGE. Crystals of the form 1 type were grown by the hanging drop method (protein: reservoir solution ratio 1:1) at 17°C using PEG 8000 (14-16%) in the presence of 25 mM MES (pH 6.2), 50 mM (NH4
and 7 mM MgCl2
as a precipitant. Crystals typically reached maximal size over 1-2 weeks. Crystals of form 2 were grown from protein containing wild type sequence, expressed and purified in the same way. Hanging drops composed of 2μl protein, 1μl NaH2
(50mM) and 2.5μl PEG 3350 (18%) were equilibrated against 900 μl of a solution containing PEG 3350 (24%) at 17°C. Again crystals reached maturity over the span of 1-2 weeks. For both forms crystals were cryoprotected using 35% PEG 8000 (form 1) and 36% PEG 3350 (form 2) prior to flash cooling.
Diffraction sets were collected as 0.5° oscillation frames on an R-AXIS IV image plate detector using CuKa radiation from a rotating anode generator operating at 50 kV and 100 mA with Rigaku Varimax optics. Data integration, merging, and scaling were performed with D*Trek suite of programs. Form 1 crystals diffracted x-rays to 1.75 Å and were of the P 21
space group, while form 2 crystals diffracted x-rays to 2.15 Å and were also of the P 21
space group. Initial phases for Form 1 were generated using the program Phaser from the Phenix suite of programs14
and required the use of a composite model generated from structures of KsgA12
(1QYR), P. falciparum
(2H1R), and human Dim1 (1ZQ9; unpublished structure from the Structural Genomics Consortium). The initial model was improved via the autobuild function in Phenix. The resulting model was refined by iterative cycles of manual rebuilding using the program Coot18
maps followed by computational refinement with Phenix and REFMAC.19
Model quality was monitored via the use of Procheck and the valdiation functions in COOT. Structure solution and refinement for form 2 were carried out using protocols identical to those employed for form 1. Crystallographic data and refinement statistics for both forms are summarized in .
Plasmids for the complementation assays
DIM1 of S. cerevisiae was amplified from pET15b-DIM1 as an XhoI-NheI fragment and subcloned into the pESC-URA vector (Stratagene) to give pESC-URA-DIM1, which results in Dim1 with an N-terminal myc epitope. This clone and all subsequent clones were confirmed by sequencing at the Nucleic Acids Research Facilities, Virginia Commonwealth University. KsgA of E. coli was amplified from pET15b-KsgA as a NotI-SpeI fragment with an exogenous NLS (nuclear localization signal - PPKKKRKV) and subcloned into the pESC-LEU vector (Stratagene) to give pESC-LEU-KsgA, which results in KsgA with an N-terminal NLS and a C-terminal FLAG epitope. Similarly, MjDim1 was amplified from pET15b-MjDim1 as a NotI-SpeI fragment with the exogenous NLS and subcloned into pESC-LEU to give pESC-LEU-MjDim1, which results in MjDim1 with an N-terminal NLS and a C-terminal FLAG epitope. KsgA was also amplified as a BglII-PacI fragment (without exogenous NLS) and subcloned into pESC-LEU to give pESC-LEU-KsgA-2, which results in KsgA with an N-terminal FLAG epitope. DIM1, KsgA, MjDim1 were also amplified as NotI-SpeI fragments (without exogenous NLS) and subcloned into pESC-LEU to give pESC-LEU-DIM1, pESC-LEU-KsgA-3, and pESC-LEU-MjDim1-2 respectively, which results in the respective protein with a C-terminal FLAG epitope. As a control DIM1 was amplified from pET15b-DIM1 as a NotI-SpeI fragment with the exogenous NLS and subcloned into pESC-LEU to give pESC-LEU-DIM1-2, which results in Dim1 with an N-terminal NLS and a C-terminal FLAG epitope.
The N-terminal and C-terminal domains of DIM1, KsgA, MjDim1 were amplified separately as NdeI-SalI and SalI-XhoI fragments. The amplicons were then digested with SalI and ligated as following. The N-terminal domain of KsgA was ligated with the C-terminal domains of DIM1 and MjDim1 to generate KD and KM chimeras respectively. The N-terminal domain of DIM1 was ligated with the C-terminal domains of KsgA and MjDim1 to generate DK and DM chimeras respectively. The N-terminal domain of MjDim1 was ligated with the C-terminal domains of DIM1 and KsgA to generate MD and MK chimeras respectively. All six chimeras were inserted into pET15b as NdeI-XhoI fragments. DK and DM were again amplified as NotI-SpeI fragments and subcloned into pESC-LEU to give pESC-LEU-DK and pESC-LEU-DM, which results in protein with C-terminal FLAG epitope. KD, KM, MD and MK were amplified as NotI-SpeI fragments (with exogenous NLS) and subcloned into pESC-LEU to give pESC-LEU-KD, pESC-LEU-KM, pESC-LEU-MD, pESC-LEU-MK, which results in protein with N-terminal NLS and C-terminal FLAG epitopes. (See Supplementary material for primer information)
In vivo assay in bacteria
E. coli cells resistant to kasugamycin were transformed with the pET15b constructs and selected on LB plates containing ampicillin. Transformed colonies were inoculated into liquid media and grown overnight. These cultures were diluted 1:25 in fresh LB containing 50 μg/mL ampicillin and grown to an OD600 of 0.7-0.8. The cultures were again diluted 1:100 in fresh LB and 3 μL of the dilution was plated onto LB/ ampicillin containing increasing amounts of kasugamycin, from 0 to 3000 μg/mL. Plates were incubated at 37°C overnight and visually inspected for colony formation.
The E85A mutation in pESC-LEU-DIM1 was generated using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) and primers 5′-CGT AGT GGC AGT AGC AAT GGA TCC CAG AAT GG-3′ and 5′-CCA TTC TGG GAT CCA TTG CTA CTG CCA CTA CG-3′ to give pESC-LEU-dim1-E85A.
Standard S. cerevisiae
growth and handling techniques were employed. Media were standard yeast extract/peptone supplemented with either glucose (YPD) or 2% galactose (YPGal), or minimal medium (0.67% YNB, appropriate amino acids, 2% galactose), all containing 200 mg/L Geneticin (G418). Transformation was carried out using the LiOAc/PEG method.20
The yeast deletion clone #21026 generated by the Saccharomyces Genome Deletion Project (http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html
) was obtained from Invitrogen (catalog no. 95400). Y-21026 is a diploid strain, derived from BY4743, that is heterozygous for DIM1
deletion (one chromosomal DIM1
copy is replaced with the KanMX4
module). BY4743 is auxotrophic for histidine, leucine and uracil and hence the genotype of Y-21026 is MAT a/α his3Δ1/his3Δ1 leu2Δ0 /leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0 /ura3Δ0 DIM1/dim1Δ0::KanMX4. This strain was transformed with pESC-URA-DIM1
and induced to sporulate. Tetrads were dissected on synthetic medium lacking uracil to maintain selection for the plasmid. Spore colonies that were Ura+
and Geneticin-resistant were tested for mating type. From these spores, a strain of the genotype MATα his3Δ1 leu2Δ0 ura3Δ0 dim1Δ0::KanMX4
), labeled as Y26hα-URA-DIM1, was selected for further transformations.
Y26hα-URA-DIM1 was transformed with a second plasmid, pESC-LEU-DIM1, pESC-LEU-DIM1-2, pESC-LEU-KsgA, pESC-LEU-KsgA-2, pESC-LEU-KsgA-3, pESC-LEU-MjDim1, pESC-LEU-MjDim1-2, or pESC-LEU(no insert) to give Y2P-DIM1, Y2P-DIM1-2, Y2P-KsgA, Y2P-KsgA-2, Y2P-KsgA-3, Y2P-MjDim1, Y2P-MjDim1-2 and Y2P-LEU respectively.
Y26hα-URA-DIM1 was also transformed with the chimera plasmids, pESC-LEU-KD, pESC-LEU-DK, pESC-LEU-MD, pESC-LEU-DM, pESC-LEU-MK, pESC-LEU-KM and the plasmid with mutant DIM1 (pESC-LEU-dim1-E85A) to give Y2P-KD, Y2P-DK, Y2P-MD, Y2P-DM, Y2P-MK, Y2P-KM and Y2P-E85A respectively.
Fluoro-orotate resistant clones were selected on minimal medium (YNB, histidine, uracil, galactose) containing 1 g/L 5-Fluoro-orotic acid (5-FOA).15
Plates were incubated for 3 days at 30°C. Y2P-DIM1, Y2P-DIM1-2, Y2P-DM, Y2P-E85A survived on 5-FOA, losing the first plasmid, pESC-URA-DIM1
, and hence these 5-FOA resistant clones with only the second plasmid were labeled Y-DIM1, Y-DIM1-2, Y-DM, and Y-E85A respectively (Table S3
in Supplementary material).
The plasmids in the yeast strains were confirmed by PCR followed by sequencing.
Cell growth comparison between Y-DIM1 and Y-E85A
Colonies of Y-DIM1 and Y-E85A were inoculated into 2ml of minimal medium and grown to an OD600
of 1.0. Dilutions (1x to 100x) of both strains were spotted on minimal media plates and incubated for 3 days at 25, 30, and 37°C and for 4 days at 18°C. For confirmation, the rRNA was isolated from both wild type and non-dimethylated 40S subunits and primer extension was carried out on both using the primer 5′-TAA TGA TCC TTC CGC A-3′, which is complementary to the very end of 18S rRNA10,21
in Supplementary material).