SASDGB6: Resistance to inhibitors of cholinesterase 8 homolog A (Ric8A) mi...

Basic information

• Entry: Database: SASBDB / ID: SASDGB6

Sample

Resistance to inhibitors of cholinesterase 8 homolog A (Ric8A) miniGi complex

• Resistance to inhibitors of cholinesterase 8 homolog A (protein), Ric8a, Bos taurus
• miniGi (protein), synthetic construct

Function / homology

Function:

G-protein alpha-subunit binding / guanyl-nucleotide exchange factor activity / G protein-coupled receptor signaling pathway / plasma membrane / cytoplasm

Domain/homology:

Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / Armadillo-like helical / Armadillo-type fold

Component:

Synembryn-A

• Biological species: Bos taurus (cattle); synthetic construct (others)
• Citation: Journal: J Biol Chem / Year: 2019; Title: Large-scale conformational rearrangement of the α5-helix of Gα subunits in complex with the guanine nucleotide exchange factor Ric8A.; Authors: Dhiraj Srivastava / Nikolai O Artemyev / ; Abstract: Resistance to inhibitors of cholinesterase 8A (Ric8A) protein is an important G protein-coupled receptor (GPCR)-independent regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor (GEF) and a chaperone. Insights into the complex between Ric8A and Gα hold the key to understanding the mechanisms underlying noncanonical activation of G-protein signaling as well as the folding of nascent Gα proteins. Here, we examined the structure of the complex of Ric8A with minimized Gα (miniGα) in solution by small-angle X-ray scattering (SAXS) and exploited the scattering profile in modeling of the Ric8A/miniGα complex by steered molecular dynamics (SMD) simulations. A small set of models of the complex featured minimal clash scores, excellent agreement with the experimental SAXS data, and a large-scale rearrangement of the signal-transducing α5-helix of Gα away from its β-sheet core. The resulting interface involved the Gα α5-helix bound to the concave surface of Ric8A and the Gα β-sheet that wraps around the C-terminal part of the Ric8A armadillo domain, leading to a severe disruption of the GDP-binding site. Further modeling of the flexible C-terminal tail of Ric8A indicated that it interacts with the effector surface of Gα. This smaller interface may enable the Ric8A-bound Gα to interact with GTP. The two-interface interaction with Gα described here distinguishes Ric8A from GPCRs and non-GPCR regulators of G-protein signaling.

Contact author

• Dhiraj Srivastava (University of Iowa, Iowa, USA)

Models

• Model #3814: ; Type: atomic / Chi-square value: 1.637; Search similar-shape structures of this assembly by Omokage search (details)
• Model #3813: ; Type: atomic / Software: (Rosetta) / Chi-square value: 1.882; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Resistance to inhibitors of cholinesterase 8 homolog A (Ric8A) miniGi complex; Specimen concentration: 10 mg/ml / Entity id: 1575 / 1912
• Buffer: Name: 20 mM Tris, 150 mM KCl, 5 % glycerol, 1 mM TCEP / pH: 8

Entity #1575

Name: Ric8a / Type: protein
Description: Resistance to inhibitors of cholinesterase 8 homolog A
Formula weight: 55.643 / Num. of mol.: 1 / Source: Bos taurus / References: UniProt: Q5E9J8
Sequence:

GHMADPRAVA DALETGEEDV VMEALRAYNR ENSQSFTFDD AQQEDRKRLA KLLVSVLEQG LPPSRRVIWL QSIRILSRDR SCLDSFTSRR SLQALACYAG ISASQGSVPE PLNMDVVLES LKCLCNLVLS SPVAQALAAE AGLVVRLAER VGLCRQSSFP HDVQFFDLRL LFLLTALRTD VRQQLFQELQ GVRLLTRALE LTLGMTEGER HPELLPPQET ERAMEILKVL FNITFDSIKR EVDEEDAALY RHLGTLLRHC VMLAAAGDRT EELHGHAVNL LGNLPVKCLD VLLTLEPHEG SLEFLGVNMD VIRVLLSFME KRLHQTHRLK ESVAPVLSVL TECARMHRPA RKFLKAQVLP PLRDVRTRPE VGELLRNKLV RLMTHLDTDV KRVAAEFLFV LCSESVPRFI KYTGYGNAAG LLAARGLMAG GRPEGQYSED EDTDTDEYKE AKASINPVTG RVEEKPPNPM EGMTEEQKEH EAMKLVNMFD KLSRH

Entity #1912

Type: protein / Description: miniGi / Formula weight: 24.54 / Num. of mol.: 1 / Source: synthetic construct
Sequence:

AMEKAAREVK LLLLGADNSG KSTIVKQMKI IHEAGEYMPM ERVKTTGIVE THFTFKDLHF KMFDVGGQRS ERKKWIHCFE DVAAIIFCVD LSDYEEMNRM HESMKLFDSI CNNKWFTDTS IILFLNKKDL FEEKIKKSPL TICYQEYAGS NTYEEAAAYI QCQFEDLNKR KDTKEIYTHF TCATDTKNVQ FVFDAVTDVI IKNNLKDCGL F

Experimental information

• Beam: Instrument name: Advanced Photon Source (APS), Argonne National Laboratory BioCAT 18ID; City: Lemont, IL / 国: USA / Type of source: X-ray synchrotron
• Detector: Name: Pilatus3 X 1M / Pixsize x: 0.172 mm

Scan

Title: Resistance to inhibitors of cholinesterase 8 homolog A (Ric8A) miniGi complex
Measurement date: Oct 27, 2018 / Storage temperature: 4 °C / Exposure time: 0.5 sec. / Unit: 1/A /

	Min	Max
Q	0.0039	0.3813

Distance distribution function P(R)

Sofotware P(R): GNOM 4.6 / Number of points: 420 /

	Min	Max
Q	0.004209	0.3813
P(R) point	1	420
R	0	107

Result

Experimental MW: 83.6 kDa / Type of curve: sec
Comments: Purified Ric8a1-492 was mixed with excess of purified miniGi and the complex was purified by size-exclusion chromatography (SEC). The complex fractions were pooled together, concentrated and additional SEC-SAXS data was collected as described above. X-ray wavelength = UNKNOWN.

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	0.00261	1.93E-6	0.0026	2.65E-6
Radius of gyration, Rg	3.28 nm	0.003	3.24 nm	0.006

	Min	Max
D	-	10.7
Guinier point	13	118