*3.1. Designing of the LUMS1 Protein and Characterization of Its Carbohydrate Binding*

A characteristic feature of HIV-1 entry inhibitor lectins is multivalent recognition through more than one carbohydrate-binding site to attain high avidity of interaction required for potent antiviral activity. MVN, however, contains one carbohydrate-binding site present in its domain-A (Figure 1a). In this design, we removed a four-residues long insertion between strands B6 and B7 in domain-A of MVN and changed the amino acid sequence of domain-B making it identical to that of domain-A and creating two carbohydrate-binding sites. The removal of the four residues corresponding to a long flexible loop could further minimize chemical heterogeneity and reduce the protein size. Since the domain-B of MVN has been reported to adopt the structure homologous to its domain-A without this insert [19], it was conceivable that the removal of these four residues may not disturb the protein folding. Subsequently, we built a homology model of the designed protein, LUMS1, using MVN as a template to obtain a preliminary idea about its structure. The resultant model exhibited a similar structure to MVN, but unlike MVN, each of the structural domains of LUMS1 contained a putative carbohydrate-binding site and two potential inter-strand disulfide linkages (Figure 1b). In order to experimentally investigate LUMS1, we produced the recombinant protein in two different forms, unlabeled and isotopically labeled with 15N.

To find out if the purified protein was folded, we recorded a two-dimensional 15NHSQC spectrum of isotopically labeled LUMS1. Well-dispersed 1H-15N correlation cross-peaks were observed indicating that protein was folded (Figure 2). In order to test the binding of LUMS1 to α(1-2)mannobiose, the minimum glycan epitope of MVN, we used the NMR chemical shift perturbation technique, as backbone 1H-15N resonances are sensitive to change in the environment resulting from the binding of a ligand [25]. In this regard, 15NHSC spectra were acquired on a sample containing 15N-lableled LUMS1 alone in solution and in the presence of increasing concentrations of α(1-2)mannobiose. On addition of α(1-2)mannobiose, cross-peaks of several amino acids in 1H-15N correlation spectra either broadened or underwent chemical shift changes, indicating the binding of the carbohydrate. Upon the addition of the carbohydrate at two equivalents of the protein molar concentration, titration appeared to be completed as no further changes in the spectra were observed upon addition of more quantities of the carbohydrate. 15NHSQC spectra of LUMS1 free and in the presence of one equivalent of α(1-2)mannobiose is shown in Figure 2 with the expansion of a cross-peak showing a stepwise change in chemical shift with the addition of one and two equivalents of carbohydrate. At one equivalent of carbohydrate, the cross-peak in the expanded region representing free and carbohydrate-bound state of LUMS1 appeared, while in the presence of two equivalents of carbohydrate, the cross-peak representing the free state of LUMS1 completely disappeared. This indicated the binding stoichiometry as 2:1 and the presence of two carbohydrate-binding sites on LUMS1.

**Figure 2.** Carbohydrate binding of LUMS1: 15NHSQC spectra of LUMS1 alone (blue) and in the presence of one equivalent of the α(1-2)mannobiose glycan (red), superimposed. Expansions of a region of spectrum containing single cross-peak in the absence (blue) and presence of one and two equivalents of α(1-2)mannobiose are shown at the top.

#### *3.2. LUMS1 Inhibits HIV-1 Cellular Entry*

We tested LUMS1 for its anti-HIV activity in pseud-typed virus-based single-round infection assay using the HXB2 strain of HIV-1 [19]. LUMS1 potently inhibited the HIV-1 entry with EC50 of 37.2 ± 4.4 nM (Figure 3) measured from its dose-response curve. For comparison, the activity of MVN was also determined as positive control, and its EC50 was measured as 8.0 ± 1.4 nM. To determine viral specificity, the activity of LUMS1 against an amphotropic virus, VSV, was tested and it was found that LUMS1 did not inhibit VSV at a concentration as high as 10 μM (Figure S2, Supplementary Material).

**Figure 3.** Human immunodeficiency virus (HIV-1) entry inhibition by LUMS1: dose-response curve showing inhibition of pseudo-typed virus, HIV-1 strain HXB2, by LUMS1 and MVN. The proteins at varying concentration was mixed with the virus at 37 ◦C followed by the addition of TZM-bl. After 48 h, cells were lysed, and percent infection was measured through luciferase activity. The assay was performed in triplicates.
