New Carbohydrate Specificity and HIV-1 Fusion Blocking Activity of the Cyanobacterial Protein MVL: NMR, ITC and Sedimentation Equilibrium Studies

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Abstract

Carbohydrate-binding proteins that bind their carbohydrate ligands with high affinity are rare and therefore of interest because they expand our understanding of carbohydrate specificity and the structural requirements that lead to high-affinity interactions. Here, we use NMR and isothermal titration calorimetry techniques to determine carbohydrate specificity and affinities for a novel cyanobacterial protein, MVL, and show that MVL binds oligomannosides such as Man6GlcNAc2 with sub-micromolar affinities. The amino acid sequence of MVL contains two homologous repeats, each comprising 54 amino acid residues. Using multi-dimensional NMR techniques, we show that MVL contains two novel carbohydrate recognition domains composed of four non-contiguous regions comprising ∼15 amino acid residues each, and that these residues make numerous intermolecular contacts with their carbohydrate ligands. NMR screening of a comprehensive panel of di-, tri-, and high-mannose oligosaccharides establish that high-affinity binding requires at least the presence of a discrete conformation presented by Manβ(1→4)GlcNAc in the context of larger oligomannosides. As shown by sedimentation equilibrium and gel-filtration experiments, MVL is a monodisperse dimer in solution, and NMR data establish that the three-dimensional structure must be symmetric. MVL inhibits HIV-1 Envelope-mediated cell fusion with an IC50 value of ∼30 nM.

Introduction

The human immunodeficiency virus (HIV) enters a cell by way of an orchestrated series of recognition events between invading virus and target host cell.1 These include binding of the surface Envelope (Env) glycoprotein gp120 to the target cell receptor CD4,2 followed by subsequent interactions between gp120 and a chemokine receptor, CXCR4 or CCR5, depending on viral tropism.3 The conformational changes brought about by this multi-protein assembly facilitates gp41-mediated membrane fusion.

Active areas of current research in pharmaceutical companies and universities include engineering Env-derived or receptor-derived peptides or proteins that can block Env-mediated fusion as well as identifying completely novel proteins that inhibit diverse strains of HIV. Depending on their size and stability, such inhibitors have the potential to be used as antivirals in vivo, or as microbicides ex vivo. Examples of engineered proteins and peptides that inhibit viral entry include those that target the pre-fusogenic form of gp41 and those that target receptor-binding sites on gp120. Examples of nanomolar gp41 inhibitors include the peptides Fuzeon™,4 C345 and N36[Mut],6 and the engineered proteins NCCG-gp41,7 N35CCG-N138 and 5-helix.9 Other than antibodies, examples of natural proteins that potently inhibit HIV-1 Env-mediated fusion include cyanovirin-N (CVN),10 scytovirin,11 and actinohivin.12 Interestingly, all of these proteins were isolated from prokaryotic organisms, with CVN and scytovirin coming from the cyanobacteria Nostoc ellipsosporum and Scytonema varium, respectively, and actinohivin from an actinomycete strain. CVN, in particular, has been studied extensively at the structural and biochemical levels, and shown to recognize a stacked conformation of α-(1→2)-linked mannobiose13., 14. or mannotriose structures.15., 16. Consequently, CVN binds with high affinity GlcNAc2Man8 D1D3 and GlcNAc2Man9,13., 17., 18. the two mammalian high-mannose oligosaccharides that present these structures in an accessible manner; and it is through these interactions that CVN potently blocks HIV-1 Env-mediated fusion.13., 18. While the mechanisms by which scytovirin and actinohivin inhibit HIV have not been elucidated fully, early studies suggest that these bacterially derived proteins may exert their antiviral activity through high-affinity, carbohydrate-mediated interactions with gp120.

Little is known about the prevalence or function of cyanobacterial carbohydrate-binding proteins. Recently, we have been engaged in both structural and functional studies of these proteins for a variety of fundamental reasons: they represent novel proteins in terms of amino acid sequence homology and three-dimensional structures, they can recognize precise carbohydrate structures and conformations with unusually high (nanomolar) affinity, and at least some of these proteins potently block HIV-1 Env-mediated fusion. Perhaps most importantly, however, complexes formed between these proteins and their carbohydrate ligands represent novel templates for protein–carbohydrate recognition, and increase our understanding of the interactions required to achieve high-affinity protein–carbohydrate binding. Here, we report on the HIV-1 fusion blocking activity of a novel carbohydrate-binding protein, MVL, which shares no detectable sequence homology with any known protein families, and demonstrate carbohydrate specificity and stoichiometry of binding through NMR chemical shift mapping experiments, and isothermal titration calorimetry (ITC) and sedimentation equilibrium experiments.

Section snippets

Background

Microcystis viridis NIES-102 is a freshwater bloom-forming cyanobacterium that was observed to have transient hemagglutinating activity when grown in the laboratory under anaerobic conditions.19 This activity was traced to a 113 amino acid residue, 13 kDa protein, termed MVL. The amino acid sequence of MVL was determined by enzymatic digestion and sequencing, allowing for cloning of the mvl gene from template total genomic M. viridis DNA. The amino acid sequence of MVL was found to comprise two

Discussion

Protein–carbohydrate interactions mediate a host of interactions that facilitate recognition and attachment among macromolecules, cells and foreign pathogens.26., 27. For this reason, molecules that block deleterious carbohydrate-mediated interactions, such as those governing viral entry or bacterial pathogenesis, have the potential to be useful therapeutics and/or mechanistic probes. In practice, however, one fundamental obstacle to realizing this concept is the intrinsically low affinities

Cells

B-SC-1 cells (American Type Culture Collection) grown in DMEM 10% (Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum) 2 mM l-glutamine and 50 mg ml−1 of gentamycin (all from Gibco BRL, Bethesda, MD) were used for all assays.

Reagents

Recombinant vaccinia viruses used in this study were obtained from the AIDS Research and Reference program, Division of AIDS, NIAID, National Institutes of Health, and include the following recombinants (donor in parentheses): vCB-32 encoding

Acknowledgements

We thank C. Broder for the CCR5-encoding recombinant vaccinia virus, I. Hamachi for Manα(1-2)Manα-containing trisaccharides, M. Clore & D. Williams for helpful discussions, and the Intramural AIDS Targeted Antiviral Program of the Office of the Director, NIH (C.A.B.) for financial support.

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      This domain organization results in a cleft that contains the CRD, which accommodates pentasaccharides through direct interactions with 4 sugar monomers. MVL has been reported to have anti-HIV-1 and HCV activities at 30–37 nM and 14–34.3 nM, respectively (Williams et al., 2005; Kachko et al., 2013; Bewley et al., 2004). Pre-incubation of HCV with Man9GlcNAc2 prevented viral inhibition by MVL in a dose dependent manner, which is consistent with MVL inhibiting enveloped viruses through the interaction with glycans of glycoproteins (Kachko et al., 2013).

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