SASDCM6: Small GTPase Rab5 conjugated with ubiquitin at K116

Basic information

• Entry: Database: SASBDB / ID: SASDCM6

Sample

Small GTPase Rab5 conjugated with ubiquitin at K116

• Monoubiquitinated Rab5 at K165 (protein), mUbRab5K165, Homo sapiens

• Biological species: Homo sapiens (human)
• Citation: Journal: Elife / Year: 2017; Title: Site-specific monoubiquitination downregulates Rab5 by disrupting effector binding and guanine nucleotide conversion.; Authors: Donghyuk Shin / Wooju Na / Ji-Hyung Lee / Gyuhee Kim / Jiseok Baek / Seok Hee Park / Cheol Yong Choi / Sangho Lee / ; Abstract: Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5, whereas GDP release and GTP loading activities were altered in mUbRab5. By contrast, mUbRab5 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.

Contact author

• Donghyuk Shin (Sungkyunkwan University, Seoul, SK)

Models

• Model #1448: ; Type: mix / Radius of dummy atoms: 1.90 A / Chi-square value: 1.145; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1449: ; Type: mix / Radius of dummy atoms: 1.90 A / Chi-square value: 1.145; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1450: ; Type: dummy / Radius of dummy atoms: 1.80 A; Comment: The lowest NSD model from damaver with 10 independent dammif runs; Chi-square value: 1.316; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Small GTPase Rab5 conjugated with ubiquitin at K116 / Specimen concentration: 0.40-2.90
• Buffer: Name: 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2 / pH: 7.5

Entity #760

Name: mUbRab5K165 / Type: protein / Description: Monoubiquitinated Rab5 at K165 / Formula weight: 32.205 / Num. of mol.: 1 / Source: Homo sapiens
Sequence:

MQIFVKTLTG KTITLEVEPS DTIENVKAKI QDKEGIPPDQ QRLIFAGKQL EDGRTLSDYN IQKESTLHLV LRLRGGMASR GATRPNGPNT GNKICQFKLV LLGESAVGKS SLVLRFVKGQ FHEFQESTIG AAFLTQTVCL DDTTVKFEIW DTAGQERYHS LAPMYYRGAQ AAIVVYDITN EESFARAKNW VKELQRQASP NIVIALSGNK ADLANKRAVD FQEAQSYADD NSLLFMETSA KTSMNVNEIF MAIAKKLPKN EPQNPGANSA RGRGVDLTEP TQPTRNQCCS N

Experimental information

• Beam: Instrument name: Pohang Accelerator Laboratory 4C / City: Pohang / 国: South Korea / Type of source: X-ray synchrotron / Wavelength: 0.124 Å / Dist. spec. to detc.: 3 mm
• Detector: Name: ADSC Quantum 315 / Type: CCD / Pixsize x: 315 mm

Scan

Title: Monoubiquitinated Rab5 at K116 / Measurement date: Nov 21, 2016 / Storage temperature: 4 °C / Cell temperature: 4 °C / Exposure time: 10 sec. / Number of frames: 6 / Unit: 1/A /

	Min	Max
Q	0.0102	0.1494

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 929 /

	Min	Max
Q	0.0104809	0.149409
P(R) point	1	929
R	0	86.56

Result

Experimental MW: 30 kDa / D max: 8.66 / Type of curve: merged

	Guinier	Guinier error	P(R)
Forward scattering, I0	0.73	0.0045	-
Radius of gyration, Rg	2.554 nm	0.042	2.653 nm