Insights from NMR Spectroscopy into the Conformational Properties of Man‐9 and Its Recognition by Two HIV Binding Proteins

Abstract

Man9GlcNAc2 (Man-9) present at the surface of HIV constitutes the binding sites of several HIV neutralizing agents and mammalian lectin DC-SIGN that is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in free state and when bound by an HIV entry inhibitor protein and define the minimum epitopes of two HIV binding proteins using NMR spectroscopy. In this regard we developed a robust expression system for production of 13C,15N-labeled glycans in mammalian cells that facilitated the implementation of 3D 13C-edited spectra to deconvolute spectral overlap allowing for the solution structure determination of Man-9. The studies reveal that Man-9 interacts with HIV-binding proteins via distinct binding epitopes and adopts divergent conformations in the bound state. In combination with molecular dynamics we find that these receptor-bound conformations are sampled by Man-9 in the free state suggesting a mechanism for diverse recognition.

Metastable HIV-1 Surface Protein Env Sensitizes Cell Membranes to Transformation and Poration by Dual-Acting Virucidal Entry Inhibitors

“…MVN binds to mannose-α(1-2)-mannose terminal glycans, including those on HIV-1 Env, with mutations Q81K and M83R further stabilizing interactions. [22][23][24]45,46 Man-α(1-2)-man-terminating glycans have been quantified on the cell surfaces of nontransfected HEK293 cells (8.4-12.8% of total glycans). 47,48 HEK293T cells stably express the SV40 large T antigen but otherwise resemble their parent 293 cell line.…”

“…22 MVN* is a recombinant variant of the original lectin microvirin (MVN), containing Q81K and M83R mutations ( Figure 1B) that enhance MNV's ability to bind to mannose-α (1-2)-mannose terminating glycans on HIV-1 Env gp120 and has been shown to inhibit HIV-1 infection. 23,24 L4 describes the flexible peptide linker (G 4 S) 4 . Trp3 is a truncation of the HIV-1 Env membrane proximal external region (MPER) sequence at the third tryptophan [HxBc numbering 664-672 (see Figure 1C)], resulting in the peptide sequence DKWASLWNW.…”

The cyanobacterial lectin, microvirin-N, enhances the specificity and sensitivity of lipoarabinomannan-based TB diagnostic tests

“…29 The discovery of MVN, a highly specific Manα-(1,2)-Man binding lectin, was reported in 2006 by Kehr et al 23 Since then, it has been investigated for anti-HIV-1 activity and as a neutralizing agent for hepatitis C viral infections. 24,30,31 To evaluate the feasibility of MVN to selectively capture ManLAM for use in next generation TB diagnostic tests, MVN was subjected to biolayer interferometry (BLI) experiments with procurable LAM variants. LAM antibodies procured from the BEI repository and FIND were also evaluated employing the same design of experiments.…”

“…2A). 30 Stability is an important parameter to the utility of proteinbased capture reagents, especially when employed in POC diagnostic tests. As a measure of stability, the binding affinity (K D ) of MVN conjugates, stored at room temp and at 4°C, was examined against H37Rv M. tb ManLAM over time.…”

Chemical and Biophysical Approaches for Complete Characterization of Lectin–Carbohydrate Interactions

“…Recently, the group of Kato and coworkers engineered a yeast cell line to produce Man-9 (Kamiya, Yamamoto, Chiba, Jigami, & Kato, 2011). For the studies described here, we developed a reliable method for purifying milligram quantities of Man-9 from recombinant gp120 produced in the presence of the A-mannosidase inhibitor kifunensine (Sastry, Bewley, & Kwong, 2015; Shahzad-Ul-Hussan et al, 2017). …”