Human ubiquilin 1, ubiquitin-like domain

Target ID:

UBQLN1

Deposition Date:

Tuesday 30th June 2009

Families:

Ubiquitin Related Biology

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

2KLC

Authors:

Doherty R, Dhe-Paganon S, Fares C, Lemak S, Gutmanas A, Garcia M, Yee A, Montelione GT, Arrowsmith CH

Site:

Toronto

2KLC

tabs

Structure Details

Ubiquilin 1 is one of the four members of the ubiquilin protein family. Ubiquilin proteins contain an N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated domain, separated by ~450aa. The central region of ubiquilin proteins contain two STI1 motifs, capable of binding to heat shock proteins. Ubiquilin proteins physically associate with proteasomes and ubiquitin ligases, and are thought to modulate protein degradation. Ubiquilin 1 specifically interacts with ubiquitin-interacting motifs (UIMs) in the proteasomal subunit S5A, ataxin-3, HSJ1a, and EPS15[1]. Ubiquilin 1 also interacts with CD47 and Gβγ, suggesting a role in integrating adhesion and signaling components of cell migration[2]. Ubiquilin 1 is expressed at low levels in quiescent cells, and increases significantly between the G1 and S phases of the cell cycle[3].

Ubiquilin 1 has also been associated with Huntington's disease, Alzheimer's disease and Parkinson's disease. It interacts with presenilin proteins, and is found in lesions associated with Alzheimer's and Parkinson's disease[4]. It has also been observed to play a key role in regulating amyloid precursor protein trafficking from intracellular compartments to the cell surface[5]. Ubiquilin also suppresses polyQ-induced protein aggregation and toxicity in cells, and in animal models of Huntington's disease[6].

The solution structure of the ubiquitin-like domain of ubiquilin 1 reveals the typical alpha/beta ubiquitin fold with an RMS deviation of 1.13 Angstroms when comparing the 79 alpha carbons of ubiquilin 3 (1YQB). Ubiquilin 1 & 3 have surface exposed lysines that are spatially conserved with those of ubiquitin K6, K11, K27, K33, and K48. However, ubiquilin proteins have more surface exposed lysines, with 9 to 11 lysines within their ubiquitin-like domain. The electrostatic surfaces of ubiquilin 1 & 3 are positively charged with a very similar charge distribution, except that ubiquilin 3 has a negatively charged patch around residue 52, where ubiquilin has an extra lysine. These differences in surface charge distribution may influence binding specificity towards binding partners, which may be revealed through structural analysis of protein complexes involving ubiquilin family members.

Materials and Methods

UBQLN1

PDB:2KLC

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:ubh29.034.112.57D12
SGC Clone Accession:ubqln1.034.112.p15Tvlic
Tag:N-terminal tag: MGSSHHHHHHSSGRENLYFQG
Host:BL21 (DE3)RIL

Construct

Prelude:
Sequence:mgsshhhhhhssgrenlyfqgPKIMKVTVKTPKEKEEFAVPENSSVQQFKEEISKRFKSHTDQLVLIFAGKILKDQDTLSQHGIHDGLTVHLVIKTQNRP
Vector:p15Tvlic

Growth

Medium:
Antibiotics:
Procedure:Transformed BL21 (DE3)RIL Bacteria were grown in M9 minimal media supplemented with biotin, thiamine, and 10 µM ZnSO₄; ¹⁵NH₄Cl and ¹³C-glucose were the sole nitrogen and carbon source. Starter cultures (50 mL in a 250 mL flasks) were prepared with media supplemented with 100 µL of glycerol stock and shaken overnight (18 hours) at 220 rpm at 37 degC. They were used to inoculate 500 mL of growth media in a modified LEX fermentation system at 37 degC to an OD600 of 1.0. Cultures were induced with 1 mM IPTG and grown at room temperature overnight (15.5 hours). Cells were harvested by centrifugation and frozen in 50 mL Falcon tubes at -80 degC

Purification

Procedure
Lysate was clarified by centrifugation for 20 min at 4 °C and the supernatant was mixed for 20 minutes at 4 °C with 2 mL settled Ni²⁺ affinity beads. Beads were batch-washed twice with 5 mL of cold Wash buffer: spun at 2000 rpm for 6 minutes, transferred to a column, and further washed with 2 mL of wash buffer. Protein was eluted with 5 mL of Elution buffer. Buffer exchange into NMR buffer and protein concentration was performed using VivaSpin concentrators with a 5,000 molecular weight cut-off at 3000 rpm, resulting in a final volume of 300 ul.

Extraction

Procedure
Frozen cell pellets from 500 ml cultures were thawed, resuspended in 25 mL Lysis buffer and lysed by sonication.
Concentration:
Ligand
MassSpec:
Crystallization:
NMR Spectroscopy:Protein samples were transferred to a 5 mm shigemi NMR tube for data collection. A series of spectra (¹³C-edited aliphatic NOESY, ¹³C-edited aromatic NOESY, ¹⁵N-edited NOESY-HSQC, ¹³C Constant Time HSQC, 3D HNCO, 3D HNCA, 3D CBCA(CO)NH, 3D HBHA(CO)NH, 3D (H)CCH-TOCSY, and 3D H(C)CH-TOCSY) were collected at 25 degC on Bruker Avance 800 MHz or 600 MHz equipped with a z-shielded gradient triple resonance cryoprobe. Chemical shifts were referenced to external DSS. All spectra were non-uniformly sampled, and were processed using the NMRPipe, NMRDraw and multidimensional decomposition software. The backbone assignments were obtained using HNCO, CBCA(CO)NH, HBHA(CO)NH, HNCA and 15N-edited NOESY-HSQC spectra. Assignments were made using FMCgui and SPARKY software. Aliphatic side chain assignments relied on H(C)CH-TOCSY, (H)CCH-TOCSY, ¹³C-edited aliphatic NOESY and ¹⁵N-edited NOESY-HSQC spectra. 36 H-N and 39 Ca-CO RDC constraints were determined using SPARKY and Pales.
Data Collection:
Data Processing:Distance restraints for structure calculations were derived from cross-peaks in ¹⁵N-edited NOESY-HSQC, ¹³C-edited aliphatic and aromatic NOESY-HSQC spectra. NOE assignment and structure calculations were performed using FMCgui and CYANA. The quality of structure calculation was assessed by NMR structure quality assessment scores (NMR PRF scores). The best 20 of 100 CYANA structures from the final cycle were selected and subjected to molecular dynamics refinement in explicit water with RDC constraints using the program CNS. The structures were inspected by PROCHECK and MolProbity using NESG validation software package PSVS.

References

Heir, R., Ablasou, C., Dumontier, E., Elliott, M., Fagotto-Kaufmann, C., Bedford, F.K. The UBL domain of PLIC-1 regulates aggresome formation. EMBO Rep. 7(12):1252-1258 (2006)
N'Diaye, E.N., Brown, E.J. The ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with G. J. Cell Biol. 163(5):1157-1165 (2003)
Ozaki, T., Hishiki, T., Toyama, Y., Yuasa, S., Nakagawara, A., Sakiyama, S. Identification of a new cellular protein that can interact specifically with DAN. DNA Cell Biol. 16(8):985-991 (1997)
Mah, A.L., Perry, G., Smith, M.A., Monteiro, M.J. Identification of ubiquilin, a novel presenilin interactor that increases presenilin protein accumulation. J. Cell Biol. 151(4):847-862 (2000)
Hiltunen, M., Lu, A., Thomas, A.V., Romano, Rm., et al. Ubiquilin 1 modulates amyloid precursor protein trafficking and abeta secretion. J. Biol. Chem. 281(43):32240-32253 (2006)
Wang, H., Lim, P.J., Yin, C., Rieckher, M., Vogel, B.E., Monteiro, M.J. Suppression of polyglutamine-induced toxicity in cell and animal models of Huntington's disease by ubiquilin. Hum. Mol. Genet. 15(6):1025-1041 (2006)