Unique epitope–antibody interactions in the intrinsically disordered proteoglycan-like domain of human carbonic anhydrase IX defined by high-resolution NMR combined with yeast surface display

Figures & data

Table 1. Sequence of the ePG-1 polypeptide containing an N-terminal methionine (M) from the bacterial expression vector and a C-terminal linker-His-tag for purification purposes.

Peptide	CA-IX residues*
ePG-1	37–136	MPQRLPRMQEDSPLGGGSSGEDDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVEAPGDPQEPQNNAHRDKEGDAAALEHHHHHHHH

*Proline (P) after the bacterial methionine is residue 37 in the amino acid sequence of full-length CA-IX and the aspartic acid (D) before the purification tag (in underlined italics) is CA-IX residue 136. In NMR assignments, these residues are labeled as Pro1 and Asp100, respectively (see Figure 2C).

Figure 1. SPR binding studies of the CA-IX mAbs and F(Ab)s to immobilized ePG-1 polypeptide. SPR sensorgrams of (a) mAb M75 and mAb 2D7, (b) mAb 11H9 and its F(Ab) fragment, and (c) mAb 12H8 and its F(Ab) fragment to the immobilized ePG-1 polypeptide. MAb 2D7, which binds to CA-IX’s catalytic domain¹⁵, and mAb M75, which binds CA-IX’s PG domain⁸ were used as a negative and positive control, respectively. MAbs were flowed at 1.23, 3.7, 11.1, and 33.3 nM whereas F(Ab)s were flowed at 1.23, 3.7, 11.1, 33.3 and 100 nM over the chip surface with immobilized ePG-1.

Three panels of SPR sensorgrams of four monoclonal antibodies (mAbs), M75, 2D7, 11H9, and 12H8 and the F(Ab) fragments of 11H9 and 12H8. Left panel (a) has the sensorgram of M75 on top with the typical fast on (loading) phase and slow off (dissociation) phase and 2D7 at the bottom with no SPR signal. Middle panel (b) compares the sensorgrams of 11H9 with a very slow off phase (top) and its F(Ab) fragment with a faster, but still slow, off phase (bottom). Right panel (c) illustrates the sensorgrams of 12H8 with a faster off phase (top) compared to 11H9 and its F(Ab) fragment with a fast phase of dissociation.

Table 2. Phenomenological kinetic values obtained by fitting a 1:1 binding model to the SPR data for the 11H9, 12H8, and M75 anti-CA-IX mAbs and the F(Ab)s of 11H9 and 12H8 to the ePG-1 and ePG-2 polypeptides.

	e-PG1			e-PG2
mAb	ka	kd	KD	ka	kd	KD
11H9	1.70E + 06	LOD^a	LOD^a	1.82E + 06	LOD^a	LOD^a
12H8	4.78E + 06	2.71E–04	5.67E–11	NB^b	NB^b	NB^b
M75	2.54E + 03	2.70E–04	1.06E–07	2.90E + 03	2.57E–04	8.91E–08
F(Ab)
11H9	4.47E + 05	1.71E–03	3.83E–09	4.60E + 05	1.68E–03	3.66E–09
12H8	1.59E + 06	7.46E–02	4.70E–08	NB^b	NB^b	NB^b

^aThese values could not be determined from SPR sensorgrams as a result of the lack of dissociation of surface-bound 11H9 mAb in the timeframe (~10 minutes) of SPR experiments (Figure 1 and SupplFig. S1).

^bNB – No binding.

Figure 2. Solution conformation of ePG-1 characterized by NMR using backbone ³J_HN,HA coupling constants, ¹⁵N{¹H} heteronuclear NOE measurements and detailed resonance assignments. (a) essentially uniform ³J_HN,HA coupling constants (of 6–7 Hz) indicate largely random coil conformations for the ePG-1 backbone. (b) Small ¹⁵N{¹H} heteronuclear NOE values with excursions to negative values are also characteristic of unfolded polypeptide chains for ePG-1. NMR data for the central region of ePG-1 could not be analyzed due to overlaps of the ¹H/¹⁵N-HSQC signals of the highly repetitive sequence PPGEEDLPGEEDLPGEEDLP. The companion ¹⁵N longitudinal (T₁) and transverse (T₂) relaxation data are shown in SupplFig. S3B and SupplFig. S3C, respectively. (c) assigned HSQC spectrum of ePG-1. The amino acid sequence of ePG-1 (Table 1) is reproduced here with Pro (P37 of the hCA-IX sequence) as the first residue for the NMR assignment numbering. The italicized black font indicates the locations of the 11 Gly residues in the sequence of ePG-1. The inset details some signal assignments of the heavily overlapping region in the HSQC spectrum. A rectangular box outlines the side-chain amide HSQC signals of Asn and Gln residues. Two Gln residues, Q2 and Q8, and two Asn residues, N91 and N92 are located at the N-terminal and C-terminal regions of ePG-1, respectively.

Plots of the 3JHN,HA coupling constants at the top (a) and the 15N{1H} heteronuclear NOE values at the bottom (b) versus the assigned residues of 15N-labeled ePG-1. These plots have an empty region in the middle of the amino acid sequence of ePG-1 due to spectral overlaps and missing asignments. Sub-Figure c shows the 1H/15N-HSQC spectrum of 15N-labeled ePG-1 annotated by the assigned residues (on the right) and on the left displays the numbered amino acid sequence of ePG-1 used for NMR assignments.

Figure 3. (a) ¹H/¹⁵N-HSQC spectrum of ePG-1 responding to 12H8-F(Ab) binding with assignments of ePG-1 signals that perturbed and disappeared (shown in black) in the 1:1 complex. The inset shows the detailed responses of the 11 Gly signals with the disappearance of the signals of G14 and G16 of free ePG-1 (black) along with the appearance of two new HSQC signals for the complex (red). Signal movements in response to 12H8 binding were assigned tentatively as indicated by arrows in the inset, producing the chemical shift perturbations for G14, G15 and Gl6 in decreasing amplitudes. (b) Gly region of ePG-1 HSQC responding to 11H9-F(Ab) binding; the left-most panels show the superposition of the complex spectra with that of free ePG-1 (black) exemplifying the response of G25 while the right panels detailing Gly signal intensity evolutions from free ePG-1 (black) to the 1:1 (red), 1:2 (blue) and 1:5 (green) complexes, respectively. (c) Ser region of the ePG-1 HSQC spectrum responding to excess 11H9-F(Ab) binding, i.e., From 1:1 (red) to 1:2 (blue) and to 1:5 (green) for the ePG-1/F(Ab) ratio, respectively; free ePG-1 spectra are shown in black.

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Select regions of the 1H/15N-HSQC spectra of free ePG-1 and in the presence of the F(Ab) fragments of 12H8 (a) and 11H9 (b and c). For (a), the spectrum of free ePG-1 is plotted in one color (black) while in another color (red) the HSQC spectrum of ePG-1 in the presence of the 12H8 F(Ab) fragment at the 1:1 molar ratio. The perturbed residues thereby appear as having black colors in the presence of F(Ab), henceforth labeled by the assigned residues of ePG-1. Also in plot (a), there is an inset showing the details of signal movements in the Gly region of the HSQC spectrum. Plot (b) displays Gly signal movements of ePG-1 with increasing concentrations of 11H9 F(Ab), from 1:0, to 1:1, to 1:2 to 1:5 for the molar rations of ePG-1 and F(Ab), respectively. Plot (c) shows details of signal movements in another HSQC spectral region of ePG-1 with the same increasing concentrations of 11H9 F(Ab), from 1:0, to 1:1, to 1:2 to 1:5. This plot outlines resonance perturbations of select Ser residues (indicated by signal labeling) of ePG-1 responding to 11H9 F(Ab) binding.

Figure 4. Amide ¹H/¹⁵N-TROSY NMR spectrum of ¹³C/¹⁵N/²H-labeled ePG-1 in a 1:1 stoichiometric complex with the 11H9-F(Ab) fragment. Well-dispersed signals are labeled with the respective residue assignments in the triple-labeled ePG-1. It should be noted that the remaining signals in the rest of this TROSY spectrum are similar in chemical shift positions to the ¹H/¹⁵N-HSQC spectra of free ePG-1 (see Fig. 2C).

Plot of a region of the TROSY spectrum of ePG-1 annotated by the assigned residues of ePG-1 in its complex with the 11H9 F(Ab). The TROSY spectrum also contains many HSQC signals distributed within a narrow band of (NH) chemical shifts from 7.6 to 8.6 ppm.

Figure 5. Epitope mapping by yeast surface display strategy. (a) Cartoon depicting the CA-IX N-terminal fragments that were expressed on the cell surface of yeast. (b) ELISA results showing the yeast-displayed CA-IX fragments binding to mAb 11H9 (red), 12H8 (blue) and M75 (gray).

Graphic illustration of CA-IX fragments studied by yeast surface display. Panel (a) shows how the displayed fragments cover the N-terminal (i.e., PG) region of CA-IX and (b) the plot of yeast ELISA of three monoclonal antibodies, 11H9, 12H8, and M75, showing the differing binding behaviors of the three antibodies to different CA-IX fragments. 11H9 has large ELISA readings for fragments 77–91, 87–101, 67-111, and 37–140; 12H8 for fragments 37–71 and 37–140; M75 with large ELISA readings for 57–71, 77–91, 37–71 and weaker, but non-negligible ELISA signals for fragments 87–101 and 107–121.

Figure 6. Fine epitope mapping using yeast surface display (YSD). (a) Summary of antibody binding of yeast-displayed 15-residue tiling fragments of the CA-IX N-terminal region, residues 37–140. The colored sequences (12H8, green; 11H9, red, M75, blue) are the fragments within CA-IX IDR that showed the highest binding to each of the indicated antibodies by yeast cell ELISA (see Fig. 5). (b) Fine epitope mapping on 15-residue fragments that were identified to bind the antibodies through N-and C-terminal truncations to determine the “minimal” epitope followed by mutagenic alanine scan. Residues in large font and bold are those that do not tolerate alanine substitution indicating their critical role in antibody binding (see text for details).

Display of the amino acid sequence of residues 37–140 along with the 12 15-aa overlapping peptide fragments, illustrating the differing epitope (binding) loci for the three antibodies, 12H8, 11H9, and M75. Panel (a) shows the binding epitope in the form of 15-residue fragments, while panel b outlines minimal epitope for each antibody identified by sequence truncation and alanine substitutions.

Figure 7. A schematic representation of the unique interactions of the 11H9 mAb with the ePG-1 polypeptide containing two epitopes. (a) the 2:1 (PG:11H9) complex is formed preferentially when the two combining sites of 11H9 are occupied by the high-affinity epitope EDLPGEED (shown in black circles), especially when the concentration of ePG-1 is higher than or equal to the 2:1 stoichiometry. With a concentration of ePG-1 at the 1:1 (PG:11H9) ratio, the same 2:1 (PG:11H9) complex is formed for 25% of the 11H9 concentration while 50% of 11H9 forms a 1:1 (PG:11H9) complex via the high-affinity epitope. The fraction of unoccupied 11H9 (25%) interacts with the low-affinity secondary epitope, e.g. DDPLGEED, exposed in the 2:1 (PG:11H9) complex (shown in pink), leading to differential HSQC signal perturbations of residues around this site (SupplFig. S4). Such a 2:1 (PG:11H9) complex can also be formed on SPR sensor chips with the ePG-1 polypeptide immobilized via its His-tag binding to surface-conjugated anti-His antibodies (see Materials and Methods), leading to the extremely slow rate of dissociation of 11H9 mAb from the sensor chip surface (Fig. 1b and SupplFig. S1B), (a) with a concentration of ePG-1 lower than the 2:1 stoichiometry, the insufficient spacing provided by ~ 22 residues between the high-affinity and low-affinity epitopes in ePG-1 favors intermolecular cross-linking or aggregation of the 1:1 PG:11H9 species, as evidenced by turbidity and precipitation in ePG-1/11H9 samples prepared at ~ 2:1 and ~ 1:1 ratios for the concentrations of ePG-1 and 11H9, respectively (see SupplFig. S4 for further details). The 2:1 (PG:11H9) complex species shown in (A) is also present in these (2:1 and 1:1) samples as detected by both ¹H/¹⁵N-HSQC (SupplFig. S4C/D/E/F) and by size-exclusion chromatography (SupplFig. S5) after removing the cross-linked and insoluble 11H9 aggregates by centrifugation.

Cartoon diagram illustrating the two possible binding modes between 11H9 and the ePG-1 polypeptide. In (a), the mAb 11H9 having two F(Ab)’s is shown to bind ePG-1 preferentially via the high-affinity binding site EDLPGEED, resulting in the 2:1 (PG:11H9) binding stoichiometry. (b) shows the cross-linking action of ePG-1 on 11H9 when their concentration ratio reaches 2:1 (PG:11H9) or below, leading to antibody aggregation.

Table

3D	Three-dimensional
CA	Carbonic anhydrase
CHO	Chinese hamster ovary cells
D2O	Deuterium oxide
EDC	1-ethyl-3-(3-dimethylaminporpyl)- carbodiimide hydrochloride
EDTA	Ethylenediaminetetraacetic acid
ELISA	Enzyme-linked immunosorbent assay
HEPES	Hydroxyethyl piperazineethanesulfonic acid
HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO, and H(NCA)NNH	heteronuclear 3D NMR experiments for backbone resonance assignments of ^15N/^Citation13C-labeled proteins
HNHA	A 3D NMR experiment to measure the three-bond JHN-HA coupling constant
HSQC	Heteronuclear single quantum coherence
IDP	intrinsically disordered protein
IDR	intrinsically disordered protein region
LB	Luria-Bertani broth
mAbs	Monoclonal antibodies
NMR	Nuclear magnetic resonance
^Citation15N-R1	^Citation15N longitudinal relaxation rate
^Citation15N-R2	^Citation15N transverse relaxation rate
^Citation15N{^Citation1H} NOE	Heteronuclear {^Citation1H}^Citation15N nuclear Overhauser effect
PBS	Phosphate-buffered saline
PG	Proteoglycan
SDS-PAGE	Sodium dodecyl sulfate – polyacrylamide gel electrophoresis
S-NHS	Sulfo-N-hydroxysuccinimide
SPR	Surface plasmon resonance
TFA	Trifluoroacetic acid
TROSY	Transverse relaxation optimized spectroscopy
TROSY-HNCACB	TROSY-based 3D NMR experiment for backbone resonance assignments of large proteins with ^15N/^Citation13C/^Citation2H triple labeling
UPLC-SEC	Ultra-high performance liquid chromatography-size exclusion chromatography
YSD	Yeast surface display

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