Human MOCS3, rhodanese-like domain

Target ID:

MOCS3

Deposition Date:

Monday 29th June 2009

Families:

Ubiquitin Related BiologyUbiquitylation - E1 Enzymes

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

3I2V

Authors:

Bacik JP, Walker JR, Dhe-Paganon S

Site:

Toronto

3I2V

tabs

Structure Details

Human MOCS3 is an E1-like enzyme that catalyzes the adenylation and processing of two ubiquitin-like proteins, MOCS2 and Urm1[1, 2]. However, unlike other E1 enzymes, which catalyze thioester bond formation, evidence has shown that MOCS3 catalyzes the formation of a thiocarboxylate at the C-terminus of MOCS2A and Urm1 following adenylation. In order to catalyze this reaction, MOCS3 contains a rhodanese-like domain (RLD) at its C-terminus, which has been shown to be active for sulfurtransferase activity. The MOCS3 enzymatic pathway has subsequently been proposed to provide sulfur units for both the biosynthesis of molybdenum cofactor (MOCS2A pathway) and modification of tRNA (Urm1 pathway)[3, 4]. The structure of MOCS3-RLD resembles that of other rhodanese-fold enzymes with a three-stranded β-sheet surrounded by six α-helices. The catalytic cysteine, Cys412, resides in the centre of a circular loop region and appears to be stabilized by a network of main chain amide nitrogens that likely account for the increased nucleophilic character of this residue. While many rhodanese enzymes have been shown to use thiosulfate as their preferred sulfur donor, thiosulfate is a much poorer substrate for MOCS3 RLD[5]. Instead, it has been shown that the protein Nfs1 specifically interacts with and transfers sulfur from L-cysteine to the MOCS3 RLD catalytic cysteine[6]. The basis for this difference in substrate specificity has been proposed to be due to the presence of an aspartate at position 417, while this residue is commonly conserved as an arginine in other rhodaneses. Preliminary analysis of the MOCS3 RLD high-resolution crystal structure has now revealed that the side chain of Asp417 comes within 4.3 Å of the catalytic cysteine, which provides further support for this hypothesis.

Materials and Methods

MOCS3

PDB: 3I2V
Entry Clone Accession: BC015939
Entry Clone Source: uba10.BC015939.MGC.AT11-D2.pCMV-SPORT6
SGC Clone Accession: uba10.335.460.046D11 (SDC046D11)
Tag: N-terminal tag: MGSSHHHHHHSSGLVPRGS
Host: BL21 (DE3)
Vector: pET28a-LIC

Sequence (with tag):
mgsshhhhhhssglvprgsSRVSVTDYKRLLDSGAFHLLLDVRPQVEVDICRLPHALHIPLKHLERRDAESLKLLKEAIWEEKQGTQEGAAVPIYVICKLGNDSQKAVKILQSLSAAQELDPLTVRDVVGGLMAWAAKIDGTFPQY

Growth

Procedure: Competent BL21 (DE3) cells (Invitrogen, C6000-03) were transformed and grown using the LEX system (Harbinger, BEC) at 37 °C in 2L bottles (VWR, 89000-242) containing 1800 ml of TB (Sigma, T0918) supplemented with 150 mM glycerol, 100 µM Kanamycin and 600 µl antifoam 204 (Sigma A-8311). When the OD600 reached a value of about 6.0, the temperature was reduced to 15 °C, and one hour later the culture was induced with 100 µM IPTG (BioShop, IPT001) and incubated overnight (16 hours) at 15 °C. Cell pellets were collected by centrifugation (12,227 xg, 20 mins), frozen in liquid nitrogen, and stored at -80 °C. To produce the SeMet derivative, the same bacterial cells were grown using M9 SeMet High-Yield growth media kit package (Medicilon, cat#) according to manufacturer's instructions.

Protein Purification

Procedure: Beads were transferred into a 25 mL Econo-Column (Bio-Rad 732-1010) and washed with 3 x 15 mL Wash buffer. Protein was eluted with 10 mL of Elution Buffer. Samples were gel-filtered (XK 16x65 packed with HighLoad Superdex 200 resin, GE Healthcare) using an AKTAxpress (GE Healthcare, 18-6645-05) at a flow rate of 1.5 mL/min Gel-filtration buffer, and 3 mL fractions were collected in 5 ml tubes. Fractions containing protein were pooled and concentrated using a 3 KDa MW cut-off concentrator (Millipore, UFC900524) at 2851 xg to a final value of 10 mg/mL. Protein yield was 16 mg per liter of bacterial culture. Coomassie-stained SDS-PAGE showed that the product was pure (Figure 2, inset (jp, please prepare thanks, see 9pdb_prototype) and mass-spectroscopy by LCMS (Agilent 1100 Series) showed that the protein had the correct molecular weight.

Extraction

Procedure: After resuspension in 30 mL Lysis buffer per liter bacterial culture, cells were lysed by sonication ice (Virtis408912, Virsonic: 10 sec, 50 % power, 10 sec rest, 10 min total sonication time per pellet). A volume of 2.0 mL settled TALON metal-affinity resin (BD Bioscience) per 40 mL lysate (Promega, V882A) was rocked with unclarified lysate for 60 minutes at 4 °C.

Concentration:10 mg/ml

Structure Determination

Crystallization: Crystals were grown at 18 degC using the hanging drop method by mixing equal volumes of protein (10 mg/ml) and Crystallization Buffer (21% PEG3350, 0.1 M HEPES pH 7.5). Suitable crystals were cryoprotected by immersion in well solution supplemented with cryoprotectant solution (9% sucrose (wt/vol), 2% glucose (wt/vol), 8% glycerol (vol/vol), 8% ethylene glycol (vol/vol)) prior to dunking and storage in liquid nitrogen.

Data Processing: Diffraction data from 2 crystals (1.5 and 1.25 Å highest resolution, collected at 0.964 and 1.072 Å λ, respectively) of the MOCS-RLD were collected at beamline 23-ID at the Advanced Photon Source (APS). All data sets were integrated and scaled using the HKL2000 program packages. The structure was solved by SAD phasing techniques using the software package PHENIX and an initial model was built using arp/warp with the 1.5 Å resolution data set. This initial model was then used for further model building and refinement using the 1.25 Å resolution data set. Iterative model building using the graphics program Coot, and the program REFMAC5 were used for initial rounds of refinement. Final rounds of refinement were performed using phenix.refine, that led to a final model with an R factor of 16.1% (Rfree 20.4%) for data between 1.25-24.3 Å.

References

Matthies, A., et al., Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans. Proc Natl Acad Sci U S A, 2004. 101(16): p. 5946-51.
Schlieker, C.D., et al., A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway. Proc Natl Acad Sci U S A, 2008. 105(47): p. 18255-60.
Schmitz, J., et al., The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins. Biochemistry, 2008. 47(24): p. 6479-89.
Leidel, S., et al., Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA. Nature, 2009. 458(7235): p. 228-32.
Krepinsky, K. and S. Leimkuhler, Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3. Major differences in substrate specificity between eukaryotic and bacterial homologs. Febs J, 2007. 274(11): p. 2778-87.
Marelja, Z., et al., A novel role for human Nfs1 in the cytoplasm: Nfs1 acts as a sulfur donor for MOCS3, a protein involved in molybdenum cofactor biosynthesis. J Biol Chem, 2008. 283(37): p. 25178-85.