SASDDJ7: Citrate-binding PAS domain from the sensor histidine kinase, CitA...

Basic information

• Entry: Database: SASBDB / ID: SASDDJ7

Sample

Citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA

• citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA (protein), CitAP-BsLA

• Citation: Journal: Sci Rep / Year: 2017; Title: A combination of mutational and computational scanning guides the design of an artificial ligand-binding controlled lipase.; Authors: Marco Kaschner / Oliver Schillinger / Timo Fettweiss / Christina Nutschel / Frank Krause / Alexander Fulton / Birgit Strodel / Andreas Stadler / Karl-Erich Jaeger / Ulrich Krauss / ; Abstract: Allostery, i.e. the control of enzyme activity by a small molecule at a location distant from the enzyme's active site, represents a mechanism essential for sustaining life. The rational design of allostery is a non-trivial task but can be achieved by fusion of a sensory domain, which responds to environmental stimuli with a change in its structure. Hereby, the site of domain fusion is difficult to predict. We here explore the possibility to rationally engineer allostery into the naturally not allosterically regulated Bacillus subtilis lipase A, by fusion of the citrate-binding sensor-domain of the CitA sensory-kinase of Klebsiella pneumoniae. The site of domain fusion was rationally determined based on whole-protein site-saturation mutagenesis data, complemented by computational evolutionary-coupling analyses. Functional assays, combined with biochemical and biophysical studies suggest a mechanism for control, similar but distinct to the one of the parent CitA protein, with citrate acting as an indirect modulator of Triton-X100 inhibition of the fusion protein. Our study demonstrates that the introduction of ligand-dependent regulatory control by domain fusion is surprisingly facile, suggesting that the catalytic mechanism of some enzymes may be evolutionary optimized in a way that it can easily be perturbed by small conformational changes.

Contact author

• Andreas Stadler (Forschungszentrum Jülich)

Models

• Model #2027: ; Type: dummy / Software: (2.5) / Radius of dummy atoms: 2.30 A / Chi-square value: 3.243601; Search similar-shape structures of this assembly by Omokage search (details)
• Model #2028: ; Type: dummy / Radius of dummy atoms: 3.25 A / Chi-square value: 3.243601; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: Citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA; Specimen concentration: 4.98 mg/ml
• Buffer: Name: 10 mM glycine buffer, 10 mM NaCl / pH: 10

Entity #1079

Name: CitAP-BsLA / Type: protein
Description: citrate-binding CitAP domain fused to lipase A of Bacillus subtilis BsLA
Formula weight: 38.495 / Num. of mol.: 2
Sequence:

MGSSHHHHHH SSGLVPRGSH MDITEERLHY QVGQRALIQA MQISAMPELV EAVQKRDLAR IKALIDPMRS FSDATYITVG DASGQRLYHV NPDEIGKSME GGDSDEALIN AKSYVSVRKG SLGSSLRGKS PIQDATGKVI GIVSVGYTIE QLENYEKLLE DSLTEITALS IEGREAEHNP VVMVHGIGGA SFNFAGIKSY LVSQGWSRDK LYAVDFWDKT GTNYNNGPVL SRFVQKVLDE TGAKKVDIVA HSMGGANTLY YIKNLDGGNK VANVVTLGGA NRLTTGKALP GTDPNQKILY TSIYSSADMI VMNYLSRLDG ARNVQIHGVG HIGLLYSSQV NSLIKEGLNG GGQNTN

Experimental information

• Beam: Instrument name: ESRF BM29 / City: Grenoble / 国: France / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.09919 Å / Dist. spec. to detc.: 2.849 mm
• Detector: Name: Pilatus 1M

Scan

Title: Citrate-binding PAS domain from the sensor histidine kinase, CitA, fused to lipase EstA
Measurement date: Feb 6, 2014 / Storage temperature: 20 °C / Cell temperature: 20 °C / Exposure time: 1 sec. / Number of frames: 10 / Unit: 1/nm /

	Min	Max
Q	0.0361	5.0117

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 463 /

	Min	Max
Q	0.185085	2.40611
P(R) point	1	463
R	0	11.81

Result

Type of curve: single_conc
Comments: The protein is a fusion of: i) CitAP - the PAS domain from Sensor histidine kinase, CitA, from Klebsiella pneumoniae; UniProt: P52687 (Amino acid range 45-177); ii) BsLA - the Lipase EstA from Bacillus subtilis 168; UniProt: I6V559 (Amino acid range 32-212) and; iii) A linker connecting CitAP with EstA - derived from the blue light photoreceptor, YtvA, from Bacillus subtilis 168; UniProt: O34627 (Amino acid range 132-147).

	Experimental	Standard	Standard error
MW	84.42 kDa	84.42 kDa	0.068

	P(R)	Guinier	Guinier error
Forward scattering, I0	85.43	84.42	0.068
Radius of gyration, Rg	3.423 nm	3.32 nm	0.06

	Min	Max
D	-	11.81
Guinier point	32	73