Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

Lactoferrin-binding protein B (LbpB) is a lipoprotein present on the surface of Neisseria that has been postulated to serve dual functions during pathogenesis in both iron acquisition from lactoferrin (Lf), and in providing protection against the cationic antimicrobial peptide lactoferricin (Lfcn). While previous studies support a dual role for LbpB, exactly how these ligands interact with LbpB has remained unknown. Here, we present the structures of LbpB from N. meningitidis and N. gonorrhoeae in complex with human holo-Lf, forming a 1:1 complex and confirmed by size-exclusion chromatography small-angle X-ray scattering. LbpB consists of N- and C-lobes with the N-lobe interacting extensively with the C-lobe of Lf. Our structures provide insight into LbpB’s preference towards holo-Lf, and our mutagenesis and binding studies show that Lf and Lfcn bind independently. Our studies provide the molecular details for how LbpB serves to capture and preserve Lf in an iron-bound state for delivery to the membrane transporter LbpA for iron piracy, and as an antimicrobial peptide sink to evade host immune defenses.


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There are seventeen species of Neisseria that colonize humans with only two being 40 pathogenic, N. meningitidis and N. gonorrhoeae (Seifert, 2019;Tone Tønjum, 2017). N. 41 meningitidis asymptomatically colonizes ~10% of the world's population, however, once 42 pathogenic can lead to meningitidis and sepsis and lead to high fatality rates in the absence of Lf has been reported. 92 In addition to its role in iron acquisition, LbpB has been shown to provide protection against

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Complex formation of Neisserial LbpB with human lactoferrin 119 For complex formation and structural studies, we recombinantly expressed and purified the 120 soluble construct of LbpB from Neisseria meningitidis (NmLbpB) (residues 20-737) and 121 Neisseria gonorrhoeae (NgLbpB) (residues 20-728) ( Figure 1B). Solid-phase binding assays 122 showed that these purified LbpB constructs were sufficient for binding holo lactoferrin (Lf) 123 ( Figure 1C). For large-scale studies, Lf was purchased and further purified using size-exclusion 124 chromatography (SEC). Complexes of LbpB with Lf were formed upon incubation of purified 125 LbpB with an excess of Lf and separated using SEC, with the complex eluting as a left-shifted 126 peak ( Figure 1D). SDS-PAGE analysis confirmed the peak contained both components and  Table S1). The calculated radius of gyration (Rg) and maximal intramolecular 140 dimension (Dmax) are both consistent with a 1:1 stoichiometric complex. Further, we used SEC-141 SAXS analysis to show that in solution, NgLbpB alone and a complex with Lf match closely to 142 those with NmLbpB (Figures 2A, B, and Table S1).

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The crystal structure of NmLbpB in complex with human lactoferrin 145 Static small-angle X-ray scattering (SAXS) analysis showed that NmLbpB alone tends to 146 aggregate in solution at higher concentrations, while the NmLbpB-Lf complex forms a 147 monodisperse stable complex (data not shown). Therefore, crystallization screening focused on 148 both Nm and NgLbpB-Lf complexes using commercial broad-matrix screens. Initial lead 149 conditions were optimized, however, diffraction quality crystals could only be grown of the  Table   154 S2). One complex was observed in the structure containing one molecule of Lf (consisting of 155 ordered N-and C-lobes) and one molecule of NmLbpB (consisting of an ordered N-lobe, but 156 partially ordered C-lobe).

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The overall structure of the NmLbpB-Lf complex has an overall L-shape ( Figure 3B). The N 158 lobe of NmLbpB is composed of residues 1-342 forming two domains, an N-terminal handle 159 domain (residues 45-173) and an eight-stranded β-barrel domain (residues 174-342). The handle 160 domain contains four anti-parallel β-strands and an α-helix between β-strand 1 and 2. The β-161 barrel domain is packed against the β-sheet of handle domain. The C-lobe of NmLbpB is 162 composed of residues 359-718 and found largely disordered in the crystal structure as evident 163 from higher B-factors for this region compared to the rest of the structure ( Figure 3C). The C-164 lobe also contains a handle domain (residues 359-540) and an eight stranded β-barrel domain 165 (residues 541-718). While the C-lobe β-barrel domain shares homology with the N-lobe β-barrel 166 domain, the C-lobe handle domain is different as it is composed of a six-stranded β sheet packed 167 against the β-barrel domain and flanked by two antiparallel β strands. Importantly, the C-lobe of 168 LbpB contains several long anionic loops, however, none of these were observed in the electron 169 density likely due to their flexibility.

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The Lf structure also consists of N-and C-lobes with each lobe further divided into two 171 subdomains termed N1 and N2 (N-lobe) and C1 and C2 (C-lobe). In the presence of iron, the   Given our crystal structure indicated that binding to Lf was exclusively along the N-lobe of 187 NmLbpB, we used solid-phase binding assays to show that indeed Lf could only interact with the 188 full length and N-lobe only constructs, but not with the C-lobe only ( Figure 3F).

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The cryoEM structure of NgLbpB in complex with human lactoferrin 191 Crystallization with NgLbpB was unsuccessful, therefore, we used cryoEM to determine the 192 structure of NgLbpB in complex with Lf to 3.65 Å resolution (Table S3). Very good density was     Table S5). The effect of these mutations on Lf binding were first tested with solid-phasing 213 binding assays ( Figure S2) and with enzyme-linked immunosorbent assays (ELISA) and 214 compared to wild type ( Figure 5B). The results showed that all of the mutations had a significant 215 effect on Lf binding with most resulting in more than ~50% reduction in binding except for   Figure 2E). Together, these data 233 support the hypothesis that Lfcn binding is distinct from Lf binding, further supporting that Lfcn 234 binding is along the C-lobe of LbpB. While we were able to co-crystallize lactoferricin (Lfcn) 235 with NmLbpB-Lf and solve the structure, the C-lobe of LbpB remained highly disordered and 236 therefore, we were unable to determine the binding interactions. Therefore, to probe the putative 237 binding site of Lfcn, we analyzed the NmLbpB-Lf structure to determine the boundaries of the C-238 lobe loops ( Figure 6A). An electrostatic surface representation of this region shows it to be 239 highly charged with at least one large strongly electronegative region along loops consisting of 240 residues 372-383, 416-418, and 445-526; a more accurate analysis of the electrostatics here is not 241 possible given the lack of order in the loops ( Figure 6B).

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Based on these loop boundaries, we made deletion mutants with each lacking one of the 243 loops of the C-lobe of NmLbpB. We then analyzed the effects of these deletions on Lfcn binding 244 in comparison to wild type using ITC analysis on a Nano ITC instrument ( Figure 6C and Table   245 S7). Our results show that Lfcn binds to wild type NmLbpB with a Kd of 3.7 µM, a ∆H of -11.4 binding. This confirms that these two loops together contribute to lactoferricin binding in LbpB, 254 which is likely mediated by the charged residues they contain. Exactly how lactoferricin binding 255 is mediated though by these loops will require additional structural studies.

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Our SEC-SAXS experiments suggested that Lf and lactoferricin had distinct, non-competing, 257 binding sites on LbpB. Therefore, we next performed ITC experiments using a Nano ITC 258 instrument to determine the binding properties of Lf to NmLbpB in the absence or presence of 259 lactoferricin. Our results show that Lf binds to NmLbpB alone with a Kd of 0.45 µM and a ∆H of 260 -15.1 kcal/mol (Table S8). Nearly identical binding parameters are observed for Lf binding to the 261 preformed NmLbpB-lactoferricin complex, indicating that the presence of lactoferricin has no 262 effect on Lf binding ( Figure 6D and Table S8). We next tested the effect of the presence of Lf on 263 lactoferricin binding. Our earlier results showed that lactoferricin binds to NmLbpB alone with a 264 Kd of 3.7 µM and a ∆H of -11.4 kcal/mol, while here we found that lactoferricin binds to the 265 NmLbpB-Lf complex with a Kd of 7.3 µM and a ∆H of -10.6 kcal/mol ( Figure 6E and Table S8).

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Only ~2-fold difference in Kd was observed here, however, no change in ∆H indicating that 267 binding of lactoferricin to NmLbpB is not disrupted in the presence of Lf.

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The role of LbpB in Neisserial pathogenesis has been debated over the years given that it 271 appears to share many properties with TbpB, which is involved in iron scavenging from 272 transferrin (Tf), but has also been shown to be important in protecting against the host      For dot blot assays with NgLbpB wild type and mutants, 10 µL of each sample was spotted 394 on activated PVDF membrane and air dried. 1x PBS with BSA (5%) was used for blocking 395 unoccupied sites followed by three washes with 1x PBST. The HRP-conjugated holo-Lf (Lf-396 HRP) probe was used to monitor Lf binding. Excess probe was removed by washes with 1x 397 PBST and 1x PBS. Finally, ECL substrate was used for detection of HRP activity.

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Size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) 400 Purified samples were subjected to in-line size exclusion chromatography coupled to small- Crysol.

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Crystallization, data collection and structure determination using X-ray crystallography 412 For crystallization, NmLbpB-Lf was concentrated to 7.4 mg/ml. Initial crystallization trials 413 were performed using commercial screens using hanging-drop vapor-diffusion method at 20°C.  Grid preparation, data collection, and structure determination using cryoEM 430 Purified NgLbpB-Lf complex was applied to Quantifoil R 3.5/1 Cu 200 grids, that were first 431 glow discharged using a Pelco EasiGlow instrument, and plunge-frozen using Vitrobot Mark IV 432 (Thermo Fisher Scientific). Grids were screened and the grid with optimal particle distribution 433 and ice-thickness was used for data collection using a Titan Krios G1 microscope equipped with