Ligand-induced structural transitions combined with paramagnetic ions facilitate unambiguous NMR assignments of methyl groups in large proteins.

NMR spectroscopy allows the study of biomolecules in close-to-native conditions. Structural information can be inferred from the NMR spectra when an assignment is available. Protein assignment is usually a time-consuming task, being specially challenging in the case of large, supramolecular systems. Here, we present an extension of existing state-of-the-art strategies for methyl group assignment that partially overcomes signal overlapping and other difficulties associated to isolated methyl groups. Our approach exploits the ability of proteins to populate two or more conformational states, allowing for unique NOE restraints in each protein conformer. The method is compatible with automated assignment algorithms, granting assignments beyond the limits of a single protein state. The approach also benefits from long-range structural restraints obtained from metal-induced pseudocontact shifts (PCS) and paramagnetic relaxation enhancements (PREs). We illustrate the method with the complete assignment of the 199 methyl groups of a MILproSVproSAT methyl-labeled sample of the UDP-glucose pyrophosphorylase enzyme from Leishmania major (LmUGP). Protozoan parasites of the genus Leishmania causes Leishmaniasis, a neglected disease affecting over 12 million people worldwide. LmUGP is responsible for the de novo biosynthesis of uridine diphosphate-glucose, a precursor in the biosynthesis of the dense surface glycocalyx involved in parasite survival and infectivity. NMR experiments with LmUGP and related enzymes have the potential to unravel new insights in the host resistance mechanisms used by Leishmania major. Our efforts will help in the development of selective and efficient drugs against Leishmania.

Assignment of Ala, Ile, LeuproS, Met, and ValproS methyl groups of the protruding domain of murine norovirus capsid protein VP1 using methyl-methyl NOEs, site directed mutagenesis, and pseudocontact shifts.

The protruding domain (P-domain) of the murine norovirus (MNV) capsid protein VP1 is essential for infection. It mediates receptor binding and attachment of neutralizing antibodies. Protein NMR studies into interactions of the P-domain with ligands will yield insights not easily available from other biophysical techniques and will extend our understanding of MNV attachment to host cells. Such studies require at least partial NMR assignments. Here, we describe the assignment of about 70% of the Ala, Ile, LeuproS, Met, and ValproS methyl groups. An unfavorable distribution of methyl group resonance signals prevents complete assignment based exclusively on 4D HMQC-NOESY-HMQC experiments, yielding assignment of only 55 out of 100 methyl groups. Therefore, we created point mutants and measured pseudo contact shifts, extending and validating assignments based on methyl-methyl NOEs. Of note, the P-domains are present in two different forms caused by an approximate equal distribution of trans- and cis-configured proline residues in position 361.

Complete assignment of Ala, Ile, LeuProS, Met and ValProS methyl groups of the protruding domain from human norovirus GII.4 Saga.

Attachment of human noroviruses to histo blood group antigens (HBGAs) is thought to be essential for infection, although how this binding event promotes infection is unknown. Recent studies have shown that 60% of all GII.4 epidemic strains may undergo a spontaneous post-translational modification (PTM) in an amino acid located adjacent to the binding pocket for HBGAs. This transformation proceeds with an estimated half-life of 1-2 days under physiological conditions, dramatically affecting HBGA recognition. The surface-exposed position of this PTM and its sequence conservation suggests a relevant role in immune escape and host-cell recognition. As a first step towards the understanding of the biological implications of this PTM at atomic resolution, we report the complete assignment of methyl resonances of a MILProSVProSA methyl-labeled sample of a 72 kDa protruding domain from a GII.4 Saga human norovirus strain. Assignments were obtained from methyl-methyl NOESY experiments combined with site-directed mutagenesis and automated assignment. This data provides the basis for a detailed characterization of the PTM-driven modulation of immune recognition in human norovirus on a molecular level.

Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl-methyl NOESY.

Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl-methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.