Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry.

Infection by the humannoroviruses (hNoV), for the vast majority of strains, requires attachment of the viral capsid to histo blood group antigens (HBGAs). The HBGA-binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for the binding of HBGAs to P dimers and, in some cases, disagreed on whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of a reference protein is crucial. We analysed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of ß-sheets has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.

Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection.

Major Histocompatibility Complex (MHC) class I molecules selectively bind peptides for presentation to cytotoxic T cells. The peptide-free state of these molecules is not well understood. Here, we characterize a disulfide-stabilized version of the human class I molecule HLA-A*02:01 that is stable in the absence of peptide and can readily exchange cognate peptides. We present X-ray crystal structures of the peptide-free state of HLA-A*02:01, together with structures that have dipeptides bound in the A and F pockets. These structural snapshots reveal that the amino acid side chains lining the binding pockets switch in a coordinated fashion between a peptide-free unlocked state and a peptide-bound locked state. Molecular dynamics simulations suggest that the opening and closing of the F pocket affects peptide ligand conformations in adjacent binding pockets. We propose that peptide binding is co-determined by synergy between the binding pockets of the MHC molecule.

Human norovirus GII.4(MI001) P dimer binds fucosylated and sialylated carbohydrates.

Human noroviruses (HuNoV), members of the family Caliciviridae, are the major cause of acute viral gastroenteritis worldwide. Successful infection is linked to the ability of the protruding (P) domain of the viral capsid to bind histo-blood group antigens (HBGA). Binding to gangliosides plays a major role for many nonhuman calici- and noroviruses. Increasing evidence points to a broader role of sialylated carbohydrates such as gangliosides in norovirus infection. Here, we compare HBGA and ganglioside binding of a GII.4 HuNoV variant (MI001), previously shown to be infectious in a HuNoV mouse model. Saturation transfer difference nuclear magnetic resonance spectroscopy, native mass spectrometry (MS) and surface plasmon resonance spectroscopy were used to characterize binding epitopes, affinities, stoichiometry and dynamics, focusing on 3'-sialyllactose, the GM3 ganglioside saccharide and B antigen. Binding was observed for 3'-sialyllactose and various HBGAs following a multistep binding process. Intrinsic affinities (Kd) of fucose, 3'-sialyllactose and B antigen were determined for the individual binding steps. Stronger affinities were observed for B antigen over 3'-sialyllactose and fucose, which bound in the mM range. Binding stoichiometry was analyzed by native MS showing the presence of four B antigens or two 3'-sialyllactose in the complex. Epitope mapping of 3'-sialyllactose revealed direct interaction of α2,3-linked sialic acid with the P domain. The ability of HuNoV to engage multiple carbohydrates emphasizes the multivalent nature of norovirus glycan-specificity. Our findings reveal direct binding of a GII.4 HuNoV P dimer to α2,3-linked sialic acid and support a broader role of ganglioside binding in norovirus infection.

Anti-trypanosomal activity of cationic N-heterocyclic carbene gold(I) complexes.

Two gold(I) N-heterocyclic carbene complexes 1a and 1b were tested for their anti-trypanosomal activity against Trypanosoma brucei parasites. Both gold compounds exhibited excellent anti-trypanosomal activity (IC50=0.9-3.0nM). The effects of the gold complexes 1a and 1b on the T. b. brucei cytoskeleton were evaluated. Rapid detachment of the flagellum from the cell body occurred after treatment with the gold complexes. In addition, a quick and complete degeneration of the parasitic cytoskeleton was induced by the gold complexes, only the microtubules of the detached flagellum remained intact. Both gold compounds 1a and 1b feature selective anti-trypanosomal agents and were distinctly more active against T. b. brucei cells than against human HeLa cells. Thus, the gold complexes 1a and 1b feature promising drug candidates for the treatment of trypanosome infections such as sleeping sickness (human African Trypanosomiasis caused by Trypanosoma brucei parasites).

Improved anticancer and antiparasitic activity of new lawsone Mannich bases.

Substituted lawsone Mannich bases 2a-e, 3a-e and 4a-e were prepared and tested for their biological activities. The new fatty alkyl substituted compounds 2a-c exhibited strong and selective growth inhibitory activities in the low one-digit micromolar and sub-micromolar range against a panel of human cancer cell lines associated with ROS formation. In addition, compounds 2a-c revealed sub-micromolar anti-trypanosomal activities against parasitic Trypanosoma brucei brucei cells via deformation of the microtubule cytoskeleton. The N-hexadecyl compound 2c was also highly active against locally isolated Entamoeba histolytica parasite samples exceeding the activity of metronidazole.

Attachment of norovirus to histo blood group antigens: a cooperative multistep process.

Human noroviruses recognize histo blood group antigens (HBGAs) as cellular attachment factors. Recently, it has been discovered that norovirus infection can be significantly enhanced by HBGA binding. Yet the attachment process and how it promotes host-cell entry is only poorly understood. The binding of a norovirus protruding (P) domain of a predominant GII.4 Saga strain to HBGAs at atomic resolution was studied. So far, independent and equivalent multiple binding sites were held responsible for attachment. Using NMR experiments we show that norovirus-HBGA binding is a cooperative multi-step process, and native mass spectrometry reveals four instead of two HBGA binding sites per P-dimer. An accompanying crystallographic study has disclosed four instead of two L-fucose binding sites per P-dimer of a related GII.10 strain1 further supporting our findings. We have uncovered a novel paradigm for norovirus-HBGA recognition that will inspire further studies into norovirus-host interactions.

A hydrolase-based reporter system to uncover the protein splicing performance of an archaeal intein.

Extein amino acid residues around the splice site junctions affect the functionality of inteins. To identify an optimal sequence context for efficient protein splicing of an intein from the thermoacidophilic archaeon Picrophilus torridus, single extein amino acid residues at the splice site junctions were continuously deleted. The construction of a set of different truncated extein variants showed that this intein tolerates multiple amino acid variations near the excision sites and exhibits full activity when -1 and +1 extein amino acid residues are conserved in an artificial GST-intein-HIS fusion construct. Moreover, splicing of the recombinant intein took place at temperatures between 4 and 42 °C with high efficiency, when produced in Escherichia coli. Therefore, structural model predictions were used to identify optimal insertion sites for the intein to be embedded within a hemicellulase from the psychrophilic bacterium Pseudoalteromonas arctica. The P. torridus intein inserted before amino acid residue Thr75 of the reporter enzyme retained catalytic activity. Moreover, the catalytic activity of the xylan-degrading hydrolase could be easily monitored in routine plate assays and in liquid test measurements at room temperature when produced in recombinant form in E. coli. This tool allows the indirect detection of the intein's catalytic activity to be used in screenings.