Fig 1.
Domain organization, thermo-stability, and structure of NHBA.
A) The constructs of NHBA studied here are shown and compared to the full-length protein. Domains are depicted as schematic boxes and labeled. NalP and Human Lactoferrin (hLF) cleavage sites are shown with dashed lines and labeled. B) DSC profiles of full-length NHBAp20 (red), NHBA133-427 (pink), and NHBA312-427 (blue). Tm values of each peak are labelled. C) The crystal structure of NHBA133-427 is shown as cartoon. D) Superposition of one monomer of the NHBA133-427 crystal structure (light and dark pink cartoon as in A) onto the one of the ten model from the NHBA NMR structure (pdb 2LFU, dark grey). A black dashed line connects common residues P295. E) Open-book view of the interface between the β-hairpin (light pink, left) and the β-barrel (dark pink, right), with hydrophobic residues that make the interface colored in orange, several Phe and Tyr side chains are shown as thick sticks.
Table 1.
Data collection and refinement statistics.
Fig 2.
Structure of the Fab5H2:NHBA312-427 complex.
A) Overall structure of the complex between Fab5H2 (H and L chain colored blue/green respectively) and the NHBA312-427 (colored in magenta with the epitope surface colored in grey). Buried surfaces at the epitope-paratope interface correspond to ~11% and ~4% of the total surface area of NHBA312-427 and Fab5H2, respectively. B) Front view of NHBA312-427 showing the five strands of its C-terminal β-barrel face that make the 5H2 epitope. The 5H2-interacting interface is colored in dark and light grey to distinguish regions contacted by 5H2 H (top) and L (bottom) chain respectively. Blue and green tubes show the 5H2 CDRs, while the rest of the Fab is omitted for clarity.
Fig 3.
Polar contacts between Fab5H2 and NHBA.
The β-barrel of NHBA, shown as dark-pink cartoon, is shown in the background in the middle of the figure. Grey surface depicts the entire 5H2 epitope, while patches of residues involved in direct bonds with Fab 5H2 are shown as solid dark-pink surfaces, and are circled and labelled. The network of polar interactions between the HC or LC of Fab 5H2 and NHBA are shown in the zoomed-in circles A) and B), respectively. C) Details of the patch III interactions, with zoom (D) into the salt bridge between NHBA R339 and Fab 5H2 D100.
Fig 4.
Thermostability of NHBA312-427 mutants and effects on 5H2 binding.
A) Cartoon representation of NHBA312-427 β-barrel and surface representation of 5H2 epitope mapped in the X-ray structure, with location of the mutated sites highlighted in green and labeled. The three spots (I, II, III) mostly involved in 5H2 recognition are boxed by dotted line. B) Tm values of NHBA mutants as studied by DSC. C) Sensorgrams profiles of the SPR experiments performed using captured Fab 5H2 and NHBA312-427 mutants. Colors corresponding to each of the mutants are reported on the left. D) Zoom into region II: comparison between the direct interactions of 5H2 with NHBA312-427 wild type (center) and predictions of the NHBA312-427 mutants R339G-K367A (right) and R339A-K367A (left).
Fig 5.
Binding-induced conformational changes in the 5H2 structures.
A) Rotamers of residues F102, Y35 and Y52 of the unbound Fab 5H2 chains HL (green), MN (orange) and OP (raspberry), compared to those of the NHBA312-427-bound Fab 5H2 chains HL (blue). B) Top view of the superposition between unbound Fab 5H2 chains HL, MN, OP and NHBA312-427-bound Fab 5H2 chains HL (blue). Region with the major differences are boxed. C) Zoom into CDR-H3 and CDR-L2 of the unbound Fab 5H2 chains OP and NHBA312-427-bound Fab5H2 chains HL, where a dotted line shows the missing atoms of the CDR-L2 from the OP chains. D) Surfaces of the triad F102-Y52-Y35 of the unbound 5H2 structures are shown on top, compared with the NHBA312-427-bound 5H2 chains HL (bottom). Rmsd values of SSM between H chain variable domains of 5H2 are reported. Chain H of 5H2-bound copy was taken as reference.
Table 2.
Comparison between the elbow angles of the three Fab copies of the unbound 5H2 and the Fab complexed with NHBA312-427.
Table 3.
Binfoding affinities between Fab5H2 and NHBA variants p2, p3 and p20 determined by Surface Plasmon Resonance.