Tonny de Beer

Molecular mechanism of NPF recognition by EH domains.

Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH–NPF interaction, the solution structures of two EH–NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I β-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the β-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.

NMR Studies of the Free α Subunit of Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone that is involved in the maintenance of the corpus luteum in early pregnancy. Glycosylation at Asn52 of its alpha subunit (alpha hCG) is essential for signal transduction, whereas the N-glycan at Asn78 stabilizes the structure of the protein. In this study, an almost complete 1H-NMR and a partial 13C-NMR spectral assignment for the amino acids and the N-glycans of alpha hCG and of an enzymatically deglycosylated form, which had a single GlcNAc residue at each of its two glycosylation sites, has been achieved. The secondary structure of alpha hCG is solution, which was determined based on NOE data, is partially similar to that of the alpha subunit in the crystal structure of hCG, but large structural differences are found for amino acid residues 33-58. In the crystal structure of hCG, residues 33-37 and 54-58 of the alpha subunit are part of an intersubunit seven-stranded beta-barrel and residues 41-47 constitute a 3(10)-helix. In contrast, in free alpha hCG in solution, amino acids 33-58 are part of a large disordered loop, indicating that in intact hCG interactions with the beta subunit of hCG stabilize the conformation of the alpha subunit. The NMR data of alpha hCG and its deglycosylated counterpart are very similar, indicating that removal of carbohydrate residues other than GlcNAc-1 does not notably affect the conformation of the protein part. However, numerous 1H-NOEs between the GlcNAc-1 residue at Asn78 and several amino acid residues show that this GlcNAc residue is tightly packed against the protein, being an integral part of the structure of the alpha subunit. 1H-NOEs across the glycosidic linkages of the glycan, resonance-line widths, and 1H and 13C chemical shifts of the other monosaccharides suggest that the remainder of the glycans at Asn78, and the glycans at Asn52 are largely extended in solution.

Rapid and simple approach for the NMR resonance assignment of the carbohydrate chains of an intact glycoprotein Application of gradient-enhanced natural abundance 1H-13C HSQC and HSQC-TOCSY to the α-subunit of human chorionic gonadotropin

The structure assessment of an intact glycoprotein in solution requires an extensive assignment of the carbohydrate NMR resonances. However, assignment of homonuelear spectra is very complicated because of the severe overlap of protein and carbohydrate signals. Application of pulsed field gradients allowed high quality natural abundance 1H‐13C HSQC and HSQC‐TOCSY spectra to be recorded of the α‐subunit of human chorionic gonadotropin. Most carbohydrate 1H‐13C correlations appear in a distinct region between the aromatic region and the protein Cα‐Hα region. The enormous reduction in overlap led to fast and unambiguous assignment of the anomeric 1H‐13C correlations. Subsequently, correlations of the monosaccharide skeleton atoms were readily assigned in the HSQC‐TOCSY spectrum.