A(I) Wilson B-factor LG268 site Rmerge Rmeas CC12 R-work R-free Variety of atoms Macromolecules Ligands Protein residues RMS bonds ( RMS angles ( Ramachandran favoredRamachandran allowedRamachandran outliersAverage B-factor Macromolecules Ligands SolventValues in parentheses are for highest resolution shell. Rmerge P pffiffiffiffi Pn n I kl I kl n i hkl P Pn j ihklI kli iI kli iSupplementary Table 1). Also, several other striking contacts are established by means of salt bridges in between Asp161 on fHbp and Arg54 around the heavy chain (Fig. 4b, upper left), and Lys185 on fHbp and Asp55Asp57 on the heavy chain (Fig. 4b, lower left), and, via hydrogen bonds involving Asn190 on fHbp and Gln101 on VH CDR3 (Fig. 4b, upper correct). Further, a water-mediated hydrogen bond is formed involving Thr91 in the light chain CDR3 and Tyr214 on fHbp (Fig. 4b, reduce right). Importantly, Asn215 on fHbp simultaneously contacts both the heavy and light chains of Fab 1A12, by hydrogen bonding together with the gamma oxygen| DOI: ten.1038s41467-018-02827-7 | www.nature.comnaturecommunicationsARTICLELIGHT CHAINNATURE COMMUNICATIONS | DOI: 10.1038s41467-018-02827-Fab 1A12 variable regionC-term N-term HEAVY CHAINfHbpFig. 2 The Fab 1A12-fHbp complicated crystal structure. Ribbon diagram in which the heavy and light chains of Fab 1A12 are colored green and yellow, respectively; fHbp is represented in cyan with a transparent surface. Artwork was ready applying PyMOLatoms of 3 serine residues (heavy chain Ser106 straight, and light chain Ser30 and Ser32 indirectly via water-mediated interactions) and with Val31 (backbone MB-0223 manufacturer nitrogen) on the light chain (Fig. 4c). A surface representation of all the fHbp residues that interact with 1A12 reveals the nature of your conformational epitope on fHbp, lying on a surface-exposed well-ordered region from the Cterminal barrel. The epitope is concentrated inside a cluster of residues targeted by the VH CDR2 and CDR3 loops, and a far more isolated region contacted by the light chain (Fig. 5a). Basis of 1A12 cross-reactivity regardless of antigenic diversity. The elucidation with the present structure makes it possible for us to provide a detailed molecular explanation for the versatility of mAb 1A12 to recognize fHbp antigens from all 3 variant groups. Remarkably, a lot of with the fHbp residues that take part in the interaction together with the Fab (12 in the 17 residues inside the 1A12 epitope) are conserved across the three distinct fHbp variants tested right here by SPR, i.e., var1.1, var2.16, and var3.45 (Fig. 5b). Notably, residues Asp161 and Asn190 are fully conserved in fHbp variants 1.1, two.16, and 3.45, and play important roles in the overall network of interactions using the Fab (Fig. 4b). Further, the motif 180KIEHLK185, and residues Asn190, Val191, and Tyr214 are also conserved inside the same 3 variants tested by SPR. Hence, the degree of conservation assigns a major role to these residues in the crossrecognition by the human mAb 1A12. The Neisseria Multi Locus Sequence Typing database now consists of 1000 various polypeptide sequences for fHbp obtained from naturally occurring strains31. For that reason, we performed a deeper evaluation in silico and calculated the degree of conservation linked with residues within the 1A12 epitope in 984 fHbp sequence variants available to date, which consist of sequences from serogroup B strains and from other serogroups31. Most notably, 5 residues (Ile181, Glu182, Leu184, Val191, and Tyr214) are 100 conserved throughout the whole fHbp sequence repertoire (Fig.