Structural Basis for Human Norovirus Capsid Binding to Bile Acids

ABSTRACT A recently developed human norovirus cell culture system revealed that the presence of bile enhanced or was an essential requirement for the growth of certain genotypes. Before this discovery, histo-blood group antigens (HBGAs) were the only well-studied cofactor known for human noroviruses, and there was evidence that several genotypes poorly bound HBGAs. Therefore, the purpose of this study was to investigate how human norovirus capsids interact with bile acids. We found that bile acids had low-micromolar affinities for GII.1, GII.10, and GII.19 capsids but did not bind GI.1, GII.3, GII.4, or GII.17. We showed that bile acid bound at a partially conserved pocket on the norovirus capsid-protruding (P) domain using X-ray crystallography. Amino acid sequence alignment and structural analysis delivered an explanation of selective bile acid binding. Intriguingly, we discovered that binding of the bile acid was the critical step to stabilize several P domain loops that optimally placed an essential amino acid side chain (Asp375) to bind HBGAs in an otherwise HBGA nonbinder (GII.1). Furthermore, bile acid enhanced HBGA binding for a known HBGA binder (GII.10). Altogether, these new data suggest that bile acid functions as a loop-stabilizing regulator and enhancer of HBGA binding for certain norovirus genotypes. IMPORTANCE Given that human norovirus virions likely interact with bile acid during a natural infection, our evidence that an HBGA nonbinder (GII.1) can be converted to an HBGA binder after bile acid binding is of major significance. Our data provide direct evidence that, like HBGAs, bile acid interaction on the capsid is an important cofactor for certain genotypes. However, more unanswered questions seem to arise from these new discoveries. For example, is there an association between the bile acid requirement and the prevalence of certain genotypes? That is, the GII.1 and GII.10 (bile acid binders) genotypes rarely caused outbreaks, whereas the GII.4 and GII.17 genotypes (bile acid nonbinders) were responsible for large epidemics. Therefore, it seems plausible that certain genotypes require bile acids, whereas others have modified their bile acid requirements on the capsid.

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Structural basis for murine norovirus engagement of bile acids and the CD300lf receptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805797115 ◽

2018 ◽

Vol 115 (39) ◽

pp. E9201-E9210 ◽

Cited By ~ 38

Author(s):

Christopher A. Nelson ◽

Craig B. Wilen ◽

Ya-Nan Dai ◽

Robert C. Orchard ◽

Arthur S. Kim ◽

...

Keyword(s):

Crystal Structure ◽

Bile Acid ◽

Bile Acids ◽

Binding Sites ◽

Structural Basis ◽

Interface Residue ◽

Cellular Receptor ◽

Murine Norovirus ◽

Bile Acid Binding ◽

P Domain

Murine norovirus (MNoV) is closely related to human norovirus (HNoV), an infectious agent responsible for acute gastroenteritis worldwide. Here we report the X-ray crystal structure of the dimeric MNoV VP1 protruding (P) domain in complex with its cellular receptor CD300lf. CD300lf binds the P domain with a 2:2 stoichiometry, engaging a cleft between the AB and DE loops of the P2 subdomain at a site that overlaps the epitopes of neutralizing antibodies. We also identify that bile acids are cofactors enhancing MNoV cell-binding and infectivity. Structures of CD300lf–P domain in complex with glycochenodeoxycholic acid (GCDCA) and lithocholic acid (LCA) reveal two bile acid binding sites at the P domain dimer interface distant from receptor binding sites. The structural determinants for receptor and bile acid binding are supported by numerous biophysical assays utilizing interface residue mutations. We find that the monomeric affinity of CD300lf for the P domain is low and is divalent cation dependent. We have also determined the crystal structure of CD300lf in complex with phosphocholine, revealing that MNoV engages its receptor in a manner mimicking host ligands including similar metal coordination. Docking of the cocomplex structures onto a cryo-EM–derived model of MNoV suggests that each virion can make multiple CD300lf engagements, and thus, infection may be driven by the avidity of cell surface clustered CD300lf. These studies identify multiple potential modulators of norovirus infection that may act to regulate the interaction between the viral capsid P domain and its cognate cellular receptor.

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Structural Basis of Nanobodies Targeting the Prototype Norovirus

Journal of Virology ◽

10.1128/jvi.02005-18 ◽

2019 ◽

Vol 93 (6) ◽

Cited By ~ 8

Author(s):

Kerstin Ruoff ◽

Turgay Kilic ◽

Jessica Devant ◽

Anna Koromyslova ◽

Alessa Ringel ◽

...

Keyword(s):

Combined Treatment ◽

Single Mode ◽

Binding Pocket ◽

Structural Basis ◽

Blood Group Antigens ◽

Combination Drug ◽

Human Norovirus ◽

X Ray Crystallography ◽

Similar Region ◽

P Domain

ABSTRACTHuman norovirus infections are a major disease burden. In this study, we analyzed three new norovirus-specific Nanobodies that interacted with the prototype human norovirus (i.e., genogroup I genotype 1 [GI.1]). We showed that the Nanobodies bound on the side (Nano-7 and Nano-62) and top (Nano-94) of the capsid-protruding (P) domain using X-ray crystallography. Nano-7 and Nano-62 bound at a similar region on the P domain, but the orientations of these two Nanobodies clashed with the shell (S) domain and neighboring P domains on intact particles. This finding suggested that the P domains on the particles should shift in order for Nano-7 and Nano-62 to bind to intact particles. Interestingly, both Nano-7 and Nano-94 were capable of blocking norovirus virus-like particles (VLPs) from binding to histo-blood group antigens (HBGAs), which are important cofactors for norovirus infection. Previously, we showed that the GI.1 HBGA pocket could be blocked with the soluble human milk oligosaccharide 2-fucosyllactose (2′FL). In the current study, we showed that a combined treatment of Nano-7 or Nano-94 with 2′FL enhanced the blocking potential with an additive (Nano-7) or synergistic (Nano-94) effect. We also found that GII Nanobodies with 2′FL also enhanced inhibition. The Nanobody inhibition likely occurred by different mechanisms, including particle aggregation or particle disassembly, whereas 2′FL blocked the HBGA binding site. Overall, these new data showed that the positive effect of the addition of 2′FL was not limited to a single mode of action of Nanobodies or to a single norovirus genogroup.IMPORTANCEThe discovery of vulnerable regions on norovirus particles is instrumental in the development of effective inhibitors, particularly for GI noroviruses that are genetically diverse. Analysis of these GI.1-specific Nanobodies has shown that similar to GII norovirus particles, the GI particles have vulnerable regions. The only known cofactor region, the HBGA binding pocket, represents the main target for inhibition. With a combination treatment, i.e., the addition of Nano-7 or Nano-94 with 2′FL, the effect of inhibition was increased. Therefore, combination drug treatments might offer a better approach to combat norovirus infections, especially since the GI genotypes are highly diverse and are continually changing the capsid landscape, and few conserved epitopes have so far been identified.

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Faculty Opinions recommendation of Structural basis for bile acid binding and activation of the nuclear receptor FXR.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014043.192544 ◽

2003 ◽

Author(s):

Matt Redinbo

Keyword(s):

Bile Acid ◽

Nuclear Receptor ◽

Structural Basis ◽

Bile Acid Binding

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Validation of Recombinant Chicken Liver Bile Acid Binding Protein as a Tool for Cholic Acid Hosting

Biomolecules ◽

10.3390/biom11050645 ◽

2021 ◽

Vol 11 (5) ◽

pp. 645

Author(s):

Giusy Tassone ◽

Maurizio Orlandini ◽

Massimo Olivucci ◽

Cecilia Pozzi

Keyword(s):

Bile Acid ◽

High Resolution ◽

Affinity Chromatography ◽

Bile Acids ◽

Drug Carriers ◽

Chicken Liver ◽

Purification Procedure ◽

Acid Binding Protein ◽

Bile Acid Binding ◽

Exogenous Substances

Bile acids (BAs) are hydroxylated steroids derived from cholesterol that act at the intestinal level to facilitate the absorption of several nutrients and also play a role as signaling molecules. In the liver of various vertebrates, the trafficking of BAs is mediated by bile acid-binding proteins (L-BABPs). The ability to host hydrophobic or amphipathic molecules makes BABPs suitable for the distribution of a variety of physiological and exogenous substances. Thus, BABPs have been proposed as drug carriers, and more recently, they have also been employed to develop innovative nanotechnology and biotechnology systems. Here, we report an efficient protocol for the production, purification, and crystallization of chicken liver BABP (cL-BABP). By means of target expression as His6-tag cL-BABP, we obtained a large amount of pure and homogeneous proteins through a simple purification procedure relying on affinity chromatography. The recombinant cL-BABP showed a raised propensity to crystallize, allowing us to obtain its structure at high resolution and, in turn, assess the structural conservation of the recombinant cL-BABP with respect to the liver-extracted protein. The results support the use of recombinant cL-BABP for the development of drug carriers, nanotechnologies, and innovative synthetic photoswitch systems.

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