Protein expression and purification
The receptor-binding domain (amino acids 1–90) of the B19V minor capsid protein, derived from isolate J35 (NIH GenBank ID AY386330.1), was cloned into pET30a vector using restriction enzymes NdeI and XhoI (New England Biolabs, Ipswich, MA, USA). The cloning resulted in the addition of amino acid residues L and E after the 90th residue of RBD due to the XhoI site. Furthermore, this construct included a C-terminal hexa-histidine tag for purification purposes and was expressed in E. coli BL21 (DE3) cells (New England Biolabs, Ipswich, MA, USA). Initially, 2 L of lysogeny broth media containing kanamycin was inoculated with 1% of pre-culture. Cells were propagated at 37°C and 200 rpm until their OD600 reached a value of 0.6 when they were harvested by centrifugation and subsequently resuspended into 1 L of minimal media containing 15NH4Cl and 13C-glucose14. The culture was incubated at 37°C and 200 rpm for 30 min before induction of protein expression for 5h by the addition of 1 mM IPTG (Thermo Fisher Scientific, Waltham, MA). Cells were then harvested and resuspended in lysis buffer containing 25 mM Tris-HCl, 500 mM NaCl, 10 mM Imidazole, pH 8, and lysed using an LM10 microfluidizer at 18,000 psi. The cell debris was removed by centrifugation at 12,000 x g for 30 min at 4°C. The clarified supernatant was applied to 0.75 mL of Ni-NTA resin (G-Biosciences, St. Louis, MO, USA) and incubated at 4°C for 1h. Following this, the resin was washed with 20 column volumes of lysis buffer and RBD was eluted with 5 column volumes of elution buffer (25 mM Tris-HCl, 500 mM NaCl, 400 mM Imidazole, pH 8). The eluent was then subjected to a second round of purification on a HiLoad 16/600 Superdex 75 pg size exclusion column (GE Healthcare, Chicago, IL, USA), pre-equilibrated in storage buffer containing 20 mM sodium phosphate buffer, pH 6.5, 50 mM NaCl, 25 mM L-arginine, and 25 mM L-glutamic acid. Sample purity was evaluated via SDS-PAGE gel with Coomassie staining. The pure protein was then concentrated using an Amicon ultra-15 centrifugal unit with a 3 KDa cutoff (Merck Millipore, Burlington, MA, USA) and stored at -20°C. Deuterium oxide (10% v/v) and 1 mM trimethylsilyl propanoic acid (TSP) were added to lock and reference the magnetic field, respectively, for NMR experiments.
Structure Prediction for the Receptor Binding Domain
To date, there is no experimentally determined structural data available for the RBD of B19V, therefore we used the AlphaFold2 structure prediction algorithm to model the RBD structure (Fig. 1). Computational predictions suggest that RBD is primarily α-helical with ~ 66% of its residues participating in α-helical secondary structural interactions. However, these α-helices are disrupted by several proline residues distributed in RBD`s primary amino acid sequence.
NMR Data Collection
All NMR spectra were acquired at 298 K using an 800 MHz/54 mm bore Bruker Avance III spectrometer, equipped with a TCI cryo-probe. An initial 1H-15N HSQC spectrum demonstrated good dispersion of the amide resonances, indicative of a well-folded protein structure. The backbone assignments were facilitated by a suite of NMR experiments, including 15N-HSQC, 13C-HSQC, HNCO, HNcaCO, HNCA, HNcoCA, HNCACB, CBCAcoNH, and HBHAcoNH (Table 1). For chemical shift referencing, 1 mM trimethylsilyl propanoic acid was employed to directly reference proton chemical shifts and indirectly reference carbon and nitrogen chemical shifts. Data acquisition and processing were conducted using Topspin versions 3.6.5 and 4.3.0, respectively.
Extent of Assignment and Data Deposition
The backbone chemical shifts of RBD were determined using CCPNMR V3.2 15. These assigned resonances were subsequently deposited in the Biological Magnetic Resonance Data Bank (BMRB) under the deposit number 52440. The 1H-15N HSQC spectrum shown in Fig. 2, indicates the backbone assignments. Within RBD amino acid sequence, three proline residues—P56, P74, and P52—were noted; however, these were not visible in the HSQC and only the CA, CB, and CO shifts for P56 and P74 could be assigned.
Similarly, other residues such as K3, W8, I40, L53, H79, and H84 lacked assignments in the HSQC but had their backbone carbon resonances assigned. Additionally, the first two residues at the amino terminus and the C-terminal His tag residues were not observed due to their dynamics. Overall, we successfully assigned ~ 99% of the backbone resonances from K3 to H90.
The chemical shifts assigned for the H, NH, CA, CB, CO, HA, and HB of the RBD domain were used as input to predict its secondary structure using Chemical Shift Indexing (CSI 3.0)16. This analysis revealed the presence of three α-helices within RBD (Fig. 3): H1 spanning E14 to V30, H2 from L35 to H44, and H3 from P56 to K71. Additionally, a turn was predicted between S48 to N51, with residues P52 to L53 predicted to be an edge β-strand, even though no β-sheet secondary structure was predicted for RBD by AlphaFold. Overall, these predictions are in good agreement with the AlphaFold structure, which indicated α-helices at positions E14-T31, D34-Y45, and P56-N72. AlphaFold also predicted short helical structures at K3-L5, P52-E54, P74-H80, S83-G85, and H90. However, the chemical shifts of these residues are more consistent with a random coil conformation (Fig. 3).