Human ubiquitin-like modifier activating enzyme 5 in complex with AMPPNP

Target ID:

UBA05

Deposition Date:

Monday 30th March 2009

Families:

Ubiquitin Related BiologyUbiquitylation - E1 Enzymes

Related Diseases:

Signalling

Structure type:

protein-ligand

Download Coordinates:

3GUC

Authors:

Walker JR, Bacik JP, Li Y, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Bochkarev A. Dhe-Paganon S.

Site:

Toronto

ICM Datapack:

ICM Datapack

3GUC

tabs

Structure Details

Conjugation of ubiquitin-like modifiers (UBLMs) to target proteins affects target protein stability, localization, regulation and interactions with other molecules. UBLMs are activated through formation of a thiol-ester bond with an E2 protein. This is catalyzed in a multistep reaction mechanism by an E1 enzyme; the enzyme 1) adenylates the C-terminal carboxyl of the UBLM, 2) forms a thioester intermediate with an active site cysteine, and 3) transfers the UBLM to a conserved cysteine of an E2. The product, E2-activated UBLM, is used by E3 ligase to covalently modify specific lysines on target substrates. Ufm1 is a UBLM that is activated by Uba5 and transferred to the Ufc1 E2 (Komatsu, M. et al., 2004). Although conserved in multicellular organisms, the biological role of Ufm1 and the Ufm-ylation pathway is unknown. The Uba5 gene contains a single Thif domain. Insertion domains found in classical E1s are either not present in Uba5 or are significantly divergent. Here we report the crystal structure of the human Ufm1 activating enzyme, Uba5, revealing a ThiF domain that is homodimerized in the canonical orientation. AMP-PNP was cocrystallized with the enzyme and appears in its canonical pocket in only one of the subunits. In the AMP-PNP bound subunit the active site cysteine appears well ordered and is located near the N-terminus of a long alpha helix. In the unbound subunit, electron density is weak or absent near the active site cysteine, suggesting greater structural flexibility in the absence of AMP-PNP. Two loops (β2-α2 and β6-α5 loops) near the active site cysteine are not modeled in this structure, presumably because of their intrinsic flexibility.

Materials and Methods

UBA5

PDB:3GUC
Entry Clone Source:uba05.BC009737.MGC.AT9-F4.pCMV-SPORT6
SGC Clone Accession:uba05.057.329.165G11 (SDC165G11)
Tag:N-terminal tag: MGSSHHHHHHSSGLVPRGS
Host:BL21 (DE3)
Vector:pET28a-LIC

Sequence (with tag):
mgsshhhhhhssglvprgsMALKRMGIVSDYEKIRTFAVAIVGVGGVGSVTAEMLTRCGIGKLLLFDYDKVELANMNRLFFQPHQAGLSKVQAAEHTLRNINPDVLFEVHNYNITTVENFQHFMDRISNGGLEEGKPVDLVLSCVDNFEARMTINTACNELGQTWMESGVSENAVSGHIQLIIPGESACFACAPPLVVAANIDEKTLKREGVCAASLPTTMGVVAGILVQNVLKFLLNFGTVSFYLGYNAMQDFFPTMSMKPNPQCDDRNCRKQQEEYKKKVAALPKQEVIQ

Growth

Procedure: Media bottles (2L) containing TB (Sigma T0918) supplemented with 1.5% glycerol, 50 µg/ml kanamycin and 600 µl antifoam 204 (Sigma A-8311) were inoculated with 50-100 ml of the overnight LB culture each. With sterilized cap/sparger (Fisher 11-138B) assemblies, bottles were placed into a circulating water bath set at 37 degC. Temperature was reduced to 15 degC one hour prior to induction at OD600 between 4 and 8 with 100 µM isopropyl-thio-B-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 degC.

Purification

Procedure: Four milliliters of TALON metal-affinity resin (BD Bioscience) was mixed for 2 hours at 4 degC with 150 mL lysate, centrifuged for 3 minutes (SX4750 rotor, Allegra X-12R, Beckman Coulter), and decanted. Beads were transferred into a 25 mL Econo-Column (Bio-Rad 732-1010) and washed with 3 x 15 mL Wash buffer. Sample was eluted with 3 column volumes of Elution buffer. Samples were gel-filtered (XK 16x65 packed with HighLoad Superdex 200 resin, GE Healthcare) using an AKTAxpress (18-6645-05, GE Healthcare) at a flow rate of 1 mL/min Gel-filtration buffer, and 3 mL fractions were collected in 5 ml tubes. Fractions containing protein were pooled and centrifuged through concentrators with 10,000 kDa cut-off (Amicon Ultra-15, UFC900524, Millipore) for 45 minutes at 3750 rpm.

Extraction

Procedure: Frozen cell pellets contained in bags (Beckman 369256), obtained from 2L cultures, were thawed by soaking in warm water, and resuspended in 25-40 mL Lysis buffer. Cell lysis was accomplished by sonication on ice (Virtis408912, Virsonic: 10 sec, 50 % power, 10 sec rest, 10 min total sonication time per pellet).
Concentration:24 mg/mL

Structure Determination

Crystallization:Crystals of Uba5 were grown at 287 K using the hanging drop method by mixing equal volumes of 0.8 M Lithium sulfate, 0.5 M Ammonium sulfate, 0.1 M Sodium Citrate pH 6.2 and 24 mg/mL protein in gel filtration buffer plus 5 mM AMPPNP and 5 mM MgCl₂. The crystals were cryoprotected by dragging the crystal through a drop containing cryoprotectant solution (9% sucrose (wt/vol), 2% glucose (wt/vol), 8% glycerol (vol/vol), 8% ethylene glycol (vol/vol)) and reservoir buffer.

References

Komatsu, M., Chiba, T., Tatsumi, K., Iemura, S., Tanida, I., Okazaki, N. et al. (2004). A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. EMBO J, 23(9), 1977-1986.