Abstract
The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 μm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.
Highlights
From the ‡Department of Computational and Structural Biology, Max F
Topological Study of LCN2-R—Sequence alignment of SLC22A17 with other transporters of the SLC22A family strongly suggests that LCN2-R adopts an unusual topology where the first 100 residues (LCN2-R-NTD) would form a soluble extracellular domain
To clarify which of these topologies is the one adopted by LCN2-R in vivo, we first performed immunofluorescence staining of CHO-K1 cells transiently transfected with rLCN2-R and immunostained for LCN2-R using antibodies against the N or C terminus as well as against the peptide sequence linking the two transmembrane domain bundles (Table 1)
Summary
Expression and Purification of hLCN2-R-NTD—The coding region for the first 105 resides of hLCN-2R (hLCN2-R-NTD) was amplified from commercial cDNA by PCR and inserted in the bacterial expression vector pET-M11, yielding pET-M11hLCN2-R-NTD, to encode hLCN2-R-NTD fused to an N-terminal His tag plus the TEV cleavage site. The protein-containing fractions were collected, and hLCN2-R-NTD was refolded via buffer exchange by dialysis against the TEV cleavage buffer (20 mM NaPi, pH 7.4, 50 mM NaCl, 0.5 mM EDTA) in the presence of 2 mM DTT. The hNGAL-containing fractions were collected, and the buffer was exchanged by dialysis to the TEV cleavage buffer (20 mM NaPi, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, 1 mM DTT). From ESI MS spectra with internal calibration using polyethylene glycol with an average molecular mass of ϳ1000 (PEG 1000), the measured mass (most abundant isotopic peak) of hLCN2-R-NTD after refolding and removal of the N-terminal His tag and the TEV cleavage site by TEV protease was 11,050.20 Da, which agrees to within 0.8 ppm with the calculated mass of the 106-residue protein with two disulfide bonds (most abundant isotopic peak, 11050.21 Da). Measurements were performed at 25 °C with an IR laser on for 30 s and off for 5 s
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