Structural analysis of nested neutralizing and non-neutralizing B cell epitopes on ricin toxin's enzymatic subunit

Abstract

In this report we describe the X-ray crystal structures of two single domain camelid antibodies (VHH), F5 and F8, each in complex with the ricin toxin's enzymatic subunit (RTA). F5 has potent toxin-neutralizing activity, while F8 has weak neutralizing activity. F5 buried a total of 1760 Å2 in complex with RTA and made contact with three prominent secondary structural elements: α–helix B (residues 98-106), β–strand h (residues 113-117), and the C-terminus of α–helix D (residues 154-156). F8 buried 1103 Å2 in complex with RTA that was centered primarily on β–strand h. As such, the structural epitope of F8 is essentially nested within that of F5. All three of the F5 complementarity determining regions (CDR) were involved in RTA contact, whereas F8 interactions were almost entirely mediated by CDR3, which essentially formed a seventh β–strand within RTA's centrally located β–sheet. A comparison of the two structures reported here to several previously reported (RTA- VHH) structures identifies putative contact sites on RTA, particularly α–helix B, associated with potent toxin-neutralizing activity. This information has implications for rational design of RTA-based subunit vaccines for biodefense.

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

“…4A). This competition profile is consistent with the structural epitope of JIV-F5 encompassing ␣-helix B, ␤-strand h, and the C-terminal portion of ␣-helix D (17). By the same token, JIY-D10 was able to bind to ricin that had been captured by GD12, PB10, PA1, SyH7, and IB2 but not WECB2, SWB1, TB12, or PH12, a profile congruent with JIY-D10's structural epitope being localized to ␣-helices D and E and ␤-strand h (Fig.…”

“…We successfully solved the X-ray crystal structures of seven of those V H Hs in complex with RTA (16)(17)(18). Screens 3 and 4 yielded an additional 16 ricin-specific V H Hs, including 7 against RTA, 1 of which was successfully crystallized (17), and 9 against RTB (Table 1).…”

“…The validity of the sandwich ELISA for epitope mapping was evaluated using the following three V H Hs whose epitopes have been resolved by X-ray crystallography (Fig. 4): JIV-F5 (PDB identifier [ID]: 4Z9K), JIY-D10 (PDB ID: 4LGR), and V1C7 (PDB ID: 5J56) (16)(17)(18). In the sandwich ELISA, the binding of JIV-F5 to ricin was affected by PB10, WECB2, and SWB1 but not by SyH7, PA1, PH12, TB12, IB2, or GD12 (Fig.…”

“…These three MAbs have overlapping epitopes on RTA's ␣-helix B, ␤-strand h, and ␣-helix D and the D-E loop (53). Moreover, the structural epitopes of five of the nine V H Hs in bin 2 have been reported (17,18). Therefore, we can assume that the remaining four V H Hs in bin 2, JIY-E3, JNM-B5, JNM-E4, and JNM-G4, contact ␣-helix B, ␤-strand h, and/or ␣-helix D ( Fig.…”

“…A collection of homo-and heterodimeric (i.e., bispecific) V H Hs were engineered with these original 20 V H Hs and shown to have enhanced avidities for ricin and, in some cases, the capacity to passively protect mice from lethal-dose ricin challenge (12)(13)(14)(15). As discussed below, the X-ray crystal structures of 10 different V H H-RTA complexes, including 7 V H Hs from the original screen (16)(17)(18), have been solved to date.…”