BMRB Entry 25799

Title:
NMR structure of human I-type lectin domain-glycan complex
Deposition date:
2015-09-07
Original release date:
2016-07-05
Authors:
Proepster, Johannes; Yang, Fan; Rabbani, Said; Ernst, Beat; Allain, Frederic; Schubert, Mario
Citation:

Citation: Proepster, Johannes; Yang, Fan; Rabbani, Said; Ernst, Beat; Allain, Frederic; Schubert, Mario. "Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8"  Proc. Natl. Acad. Sci. U. S. A. 113, 4170-4179 (2016).
PubMed: 27357658

Assembly members:

Assembly members:
Siglec-8, polymer, 145 residues, 16665.598 Da.
SUGAR_(5-MER), non-polymer, 975.920 Da.

Natural source:

Natural source:   Common Name: Human   Taxonomy ID: 9606   Superkingdom: Eukaryota   Kingdom: Metazoa   Genus/species: Homo sapiens

Experimental source:

Experimental source:   Production method: recombinant technology   Host organism: Escherichia coli   Vector: pET-43.1a

Data typeCount
13C chemical shifts623
15N chemical shifts154
1H chemical shifts1042

Additional metadata:

  • Assembly
  • Samples and Experiments
  • Software
  • Spectrometers
  • Hide all

Assembly:

Entity Assembly IDEntity NameEntity ID
1entity_11
2SUGAR (5-MER)2

Entities:

Entity 1, entity_1 145 residues - 16665.598 Da.

Construct includes Siglec-8 residues M1-H139 (carrying a C26S point mutation to enable soluble expression; numbering corresponds to mature protein), followed by six non-native C-terminal residues G140-R145 (remainder of the thrombin cleavage site after affinity-tag removal)

1   METGLUGLYASPARGGLNTYRGLYASPGLY
2   TYRLEULEUGLNVALGLNGLULEUVALTHR
3   VALGLNGLUGLYLEUSERVALHISVALPRO
4   CYSSERPHESERTYRPROGLNASPGLYTRP
5   THRASPSERASPPROVALHISGLYTYRTRP
6   PHEARGALAGLYASPARGPROTYRGLNASP
7   ALAPROVALALATHRASNASNPROASPARG
8   GLUVALGLNALAGLUTHRGLNGLYARGPHE
9   GLNLEULEUGLYASPILETRPSERASNASP
10   CYSSERLEUSERILEARGASPALAARGLYS
11   ARGASPLYSGLYSERTYRPHEPHEARGLEU
12   GLUARGGLYSERMETLYSTRPSERTYRLYS
13   SERGLNLEUASNTYRLYSTHRLYSGLNLEU
14   SERVALPHEVALTHRALALEUTHRHISGLY
15   SERLEUVALPROARG

Entity 2, SUGAR (5-MER) 5 residues - 975.920 Da.

Neu5Ac_alpha2-3[6O-SO3-]Gal_beta1-4(Fuc_alpha1-3)GlcNAc_beta_spacer //Ligand = 6-"prime"-sulfo-sialyl Lewis X tetrasaccharide with artificial chemical linker/spacer (SP) (attached to NAG C1-atom)

1   0D8NAGFUCG6SSIA

Samples:

sample_1: Siglec-8, [U-100% 15N], 0.7 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; SUGAR (5-MER)0.7 – 2.4 mM; D2O, [U-2H], 5%; H2O 95%

sample_2: Siglec-8, [U-100% 13C; U-100% 15N], 0.3 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; SUGAR (5-MER)0.7 – 2.4 mM; D2O, [U-2H], 5%; H2O 95%

sample_3: Siglec-8, [U-100% 13C; U-100% 15N], 0.3 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; SUGAR (5-MER)0.7 – 2.4 mM; D2O, [U-2H], 100%

sample_4: Siglec-8, [U-100% 15N], 0.3 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; SUGAR (5-MER)0.7 – 2.4 mM; D2O, [U-2H], 100%

sample_conditions1: ionic strength: 0.092 M; pH: 7.4; pressure: 1 atm; temperature: 293 K

sample_conditions2: ionic strength: 0.092 M; pH: 7.4; pressure: 1 atm; temperature: 303 K

sample_conditions1-2: ionic strength: 0.092 M; pH: 7.4; pressure: 1 atm

Experiments:

NameSampleSample stateSample conditions
2D 1H-15N HSQCsample_1isotropicsample_conditions1-2
2D 1H-13C HSQC aliphaticsample_2isotropicsample_conditions1-2
2D 1H-13C HSQC aromaticsample_2isotropicsample_conditions1-2
3D HNCAsample_2isotropicsample_conditions1
3D HNCACBsample_2isotropicsample_conditions1
3D CBCA(CO)NHsample_2isotropicsample_conditions1
3D HNCOsample_2isotropicsample_conditions1
3D HN(CO)CAsample_2isotropicsample_conditions1
3D HCCH-TOCSYsample_3isotropicsample_conditions1
3D 1H-15N NOESYsample_2isotropicsample_conditions1
2D 1H-1H NOESYsample_4isotropicsample_conditions1-2
3D 1H-13C NOESY aliphaticsample_2isotropicsample_conditions1
3D 1H-13C NOESY aromaticsample_2isotropicsample_conditions1
2D 1H-1H TOCSYsample_4isotropicsample_conditions1
2D 13C F2-filtered NOESYsample_3isotropicsample_conditions1-2
2D 13C F1-filtered TOCSYsample_3isotropicsample_conditions1
2D 13C F1-filtered TOCSYsample_2isotropicsample_conditions1
2D 13C F1-filtered F2-filtered NOESYsample_3isotropicsample_conditions2
3D 13C F1-edited F3-filtered NOESYsample_3isotropicsample_conditions2
2D 13C/15N F2-filtered NOESYsample_1isotropicsample_conditions2

Software:

TOPSPIN v3.0, Bruker Biospin - collection, data analysis, processing

SPARKY v3.114, Goddard - chemical shift assignment, data analysis

ATNOS/CANDID v2.1, Herrmann, Guntert and Wuthrich - peak picking

CYANA v3.0, Guntert, Mumenthaler and Wuthrich - structure calculation

TALOS+, Cornilescu, Delaglio and Bax - protein backbone torsion angle prediction from NMR chemical shifts

ProcheckNMR vv.3.5.4, Laskowski and MacArthur - validation

AMBER v12, Case, Darden, Cheatham, III, Simmerling, Wang, Duke, Luo, ... and Kollman - refinement

NMR spectrometers:

  • Bruker Avance 500 MHz
  • Bruker Avance 600 MHz
  • Bruker Avance 700 MHz
  • Bruker Avance 750 MHz
  • Bruker Avance 900 MHz

Related Database Links:

NCBI NM_014442.2
GB BC053319.1
UniProt Q9NYZ4
AlphaFold Q7Z728

Download HSQC peak lists in one of the following formats:
CSV: Backbone or all simulated peaks
SPARKY: Backbone or all simulated peaks