A(I) Wilson B-factor Rmerge Rmeas CC12 R-work R-free Number 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, numerous other striking contacts are established via salt bridges 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, by means of hydrogen bonds between Asn190 on fHbp and Gln101 on VH CDR3 (Fig. 4b, upper proper). Further, a water-mediated hydrogen bond is formed between Thr91 inside the light chain CDR3 and Tyr214 on fHbp (Fig. 4b, reduced right). Importantly, Asn215 on fHbp simultaneously contacts both the heavy and light chains of Fab 1A12, by hydrogen bonding with all the gamma oxygen| DOI: ten.1038s41467-018-02827-7 | www.nature.comnaturecommunicationsARTICLELIGHT CHAINNATURE COMMUNICATIONS | DOI: ten.1038s41467-018-02827-Fab 1A12 variable regionC-term N-term HEAVY CHAINfHbpFig. two 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 using a transparent surface. Artwork was prepared using PyMOLatoms of 3 serine residues (heavy chain Ser106 straight, and light chain Ser30 and Ser32 indirectly through water-mediated interactions) and with Val31 (backbone nitrogen) on the light chain (Fig. 4c). A surface representation of all of the fHbp residues that interact with 1A12 reveals the nature of the conformational epitope on fHbp, lying on a surface-exposed well-ordered region on the Cterminal barrel. The epitope is concentrated inside a cluster of residues targeted by the VH CDR2 and CDR3 loops, and also a additional isolated area contacted by the light chain (Fig. 5a). Basis of 1A12 cross-reactivity regardless of antigenic diversity. The elucidation on the present structure permits us to supply a detailed molecular explanation for the versatility of mAb 1A12 to recognize fHbp antigens from all 3 variant groups. Remarkably, several with the fHbp residues that participate in the interaction using the Fab (12 with the 17 residues inside the 1A12 epitope) are conserved across the three different fHbp variants Benzylideneacetone Description tested here by SPR, i.e., var1.1, var2.16, and var3.45 (Fig. 5b). Notably, residues Asp161 and Asn190 are totally conserved in fHbp variants 1.1, 2.16, and 3.45, and play key roles inside the overall network of Disodium 5′-inosinate Cancer interactions with all the Fab (Fig. 4b). Additional, the motif 180KIEHLK185, and residues Asn190, Val191, and Tyr214 are also conserved inside the same three variants tested by SPR. Consequently, the degree of conservation assigns a leading function to these residues inside the crossrecognition by the human mAb 1A12. The Neisseria Multi Locus Sequence Typing database now includes 1000 distinct polypeptide sequences for fHbp obtained from naturally occurring strains31. Therefore, we performed a deeper analysis in silico and calculated the degree of conservation connected with residues in the 1A12 epitope in 984 fHbp sequence variants accessible to date, which contain 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.