Structure, electrostatic surface, and superposition of the backbone heavy atoms for the 20 lowest energy structures of LP2086-B01. A, ribbon representation of LP2086-B01 structure. B, overlay of the structured residues for the C-domain; r.m.s. deviation 0.61 Å (the structured regions span the following residues: β11, 155-164; β12, 171-176; β13, 181-188; β14, 197-206; β15, 212-220; β16, 224-233; β17, 239-249; β18, 252-261. The hydrophobic core in the C-domain comprises residues Tyr¹⁵⁸, Tyr¹⁷⁴, Tyr²⁰³, Tyr²²⁸, Ala¹⁵⁶, Tyr¹⁵⁸, Ala¹⁶², Leu¹⁷², Tyr¹⁷⁴, Ile¹⁷⁶, Phe¹⁷⁸, Ile¹⁸⁷, Val¹⁹⁷, Leu¹⁹⁹, Val²⁰¹, Tyr²⁰³, Ala²¹², Ile²¹⁴, Val²¹⁸, Tyr²²⁰, Tyr²²⁸, Leu²³⁰, Ile²³², Val²⁴⁰, Ala²⁴⁴, Val²⁴⁶, Ile²⁵⁵, Leu²⁵⁷, Ala²⁵⁹). C, overlay of the structured residues for the N-domain; r.m.s. deviation 0.77 Å (the structured regions span the following residues: α1, 21-28; β1, 39-41; β2, 51-55; β3, 59-63; β4, 67-69; β5, 81-87; β6, 97-105; β7, 111-116; β8, 119-121; β9, 128-131; β10, 133-139. The hydrophobic core in the N-domain comprises residues Val¹⁰⁴, Leu¹¹⁵, Leu⁶⁹, Leu⁵⁴, Leu⁵², Leu⁴², Leu⁴⁰, Leu²⁶, and Phe¹⁰², Phe⁸⁴, Phe⁸², Tyr⁶³). D, overlay of the structured residues for both domains excluding the interdomain linker; r.m.s. deviation 0.96 Å. b-d, the unfolded N-terminal chain (residues 1-16) has been excluded for purposes of clarity. E, electrostatic surface representation for the entire LP2086-B01. Blue, positively charged residues; red, negatively charged residues; light gray, uncharged residues.

Flow cytometry analysis and peptide binding competition to LP2086-B01 bacterial cells. A, binding of the broad-spectrum monoclonal MN86-1075-6 raised against LP2086-B01 is inhibited by a peptide spanning sequence Phe⁸²-Glu¹⁰¹. B, subfamily B-specific monoclonal MN86-440-18 raised against 8529 has reactivity for LP2086-B01 meningococcal cells but is not competed by a peptide spanning the sequence Phe⁸²-Glu¹⁰¹. C, monoclonal antibodies MN86-813 and MN86-626-15, which cross-react with rP2086 from both subfamilies, do not recognize LP2086-B01 on the bacterial surface. Mouse IgG and the broad-spectrum monoclonal MN86-994-11 are used as negative and positive control, respectively.

Mapping of the signature residues as derived by aligning 172 known LP2086 sequences. Red, invariant residues; green, subdomain-defining residues; yellow, subfamily-defining residues. A and D, subfamily-defining residues (yellow) populate one face of the protein, involving both domains. Many of these residues are located at the C-domain and alternate the conserved (red) hydrophobic residues that form the β-barrel hydrophobic core. B and C and E and F, on the opposite side of the protein, the subdomain-defining residues (green) form two separate patches on the two domains: on the N-domain, loops Glu⁴³-Thr⁵¹ and Tyr⁶³-Gly⁶⁶ and residues on strands β2, β3, and β5; on the C-domain, neighboring residues on strands β11-β14. A and E, the MN86-1075-6 and MN86-440-18 binding regions are indicated in purple and blue, respectively. The MN86-440-18 recognizes a discontinuous epitope, which extends into regions containing subfamily-defining and subdomain-defining signature residues. MN86-1075-6 maps at a linear sequence comprising the loop Glu⁸⁹-Gln⁹³ separating strands β5 and β6, whereas MN86-440-18 induces line broadening at residues Leu⁹⁴, Glu⁸⁹, Gly⁵⁰, Glu¹²⁴, and His¹²⁵, forming a patch on neighboring strands. In all models, the unfolded N-terminal chain (residues 1-16) has been excluded.

N-domain groove and interdomain hydrophobic patches. A, groove formed between strands β1 and β10 on the N-domain of LP2086-B01. Several charged residues protect the hydrophobic core from solvent exposure. B, electrostatic representation of the N- and C-domains separately showing the hydrophobic patches involved in the interaction between the two domains.

Binding epitope of MN86-440-18 displayed on the surface of LP2086-B01. A, at an antigen/Fab ratio of 10:1, the most affected residues are Leu⁹⁴, Glu⁸⁹, and Gly⁵⁰. These residues form a small patch on one face of the N-domain. At higher concentrations of antibody (antigen/Fab ratio of 5:1), residues Glu¹²⁴ and His¹²⁵ on the nearby loop are also affected substantially. The reported values are the summation over a total of nine experiments conducted on three identical samples. Positive values of Δ (red) and negative values of Δ (green) correspond to residues experiencing above average and below average line broadening upon binding. Residues in white either have a value of Δ between the cut-offs of -1.0 and 1.0 or are overlapped. B, the position of the KDN, a common insertion in the A subfamily, is indicated. In this region of the protein, strands β3 and β4, which separate the type II turn, and the hydrophobic face of α1 between the two strands experience above average line broadening upon binding with the Fab fragment 440-18.

Proposed model representing LP2086-B01 in the bacterial membrane. The flexible linker between residues 1 and 19 allows the protein to span the LOS layer of the outer membrane. Subfamily-defining residues (yellow) populate one region on one face of the protein oriented toward the extracellular space. In violet, the linear epitopes for the monoclonals MN86-626-15 and MN86-813 are oriented toward the membrane surface and are not accessible by these mAbs in flow cytometry experiments. In green, the binding epitopes for the monoclonals MN86-440-18 and MN86-1075-6 are also exposed to the extracellular space. A single LOS molecule is indicated in red in the model. The carbohydrate torsional angles have been manually set to achieve maximum extension of the headgroup.