Hydrogen/deuterium exchange mass spectrometry and computational modeling reveal a discontinuous epitope of an antibody/TL1A Interaction

Figures & data

Figure 1. HDX kinetics mapped onto TL1A trimer crystal structure (PDB code: 2RE9). The structure only represents the extracellular domain. Regions were colored from blue to red based on the extent of deuterium uptake (%).

Figure 2. Differential HDX of TL1A upon (a) mAb1 binding and (b) Fab1 binding. Accumulated HDX reduction of peptic peptides across different exchange time periods: 30 sec (orange), 5 min (red), 20 min (light blue), 60 min (dark blue), and 240 min (black). The total levels of HDX reduction were color coded in three categories: D uptake difference 2.5–4.5 D (transparent green); D uptake difference 4.5–10 D (transparent yellow); D uptake difference >10 D (transparent red). (c) HDX kinetics curves of ⁸⁵QVYAPLRADGDKPRAHL¹⁰¹, ¹⁰²TVVRQTPTQHFKNQF¹¹⁶ and ¹⁶⁶EIRQAGRPNKPDSIT¹⁸⁰. TL1A upon mAb1 binding (blue) showed significant HDX reduction compared to TL1A alone (red). The non-deuterated peptide mass is shown in the upper left-hand corner of each plot.

Figure 3. HDX revealed two major epitope regions of mAb1, peptide 1 and peptide 2. (a) Epitope mapped onto the TL1A protein sequence. The TL1A construct is composed of the His-tagged isoleucine-zipper motif followed by the extracellular domain; (b) TL1A trimer crystal structure (PDB code: 2RE9); and (c) TL1A/DcR3 crystal structure (PDB code: 3K51).

Table 1. Computational assessment of exposed amino acids corresponding to peptide region 1. Residues that are solvent exposed and prone to protein-protein interactions are considered functional residues.

Peptide region 1
Residues	Percent Side Chain Exposure (%)	Functional Residues
E166	73.8	*
I167	41.6
R168	68.9	*
Q169	97.1	*
A170	66.1
G171	87.9
R172	51.7	*
P173	61.2
N174	18.8
K175	78.7	*
P176	44.2
D177	43.7
S178	53.6
I179	0.8
T180	7.6

Table 2. Computational assessment of exposed amino acids corresponding to peptide region 2. Residues that are solvent exposed and prone to protein-protein interactions are considered functional residues.

Peptide region 2
Residues	Percent Side Chain Exposure (%)	Functional Residues
T107	60.6
P108	81.5
T109	63.4	*
Q110	65.5	*
H111	76.9	*
F112	70.3	*
K113	63	*
N114	93.3	*
Q115	65.6	*
F116	39.2
P117	28.7
A118	19.8
L119	0.3
H120	19

Figure 4. Molecular docking of TL1A with Fab. Epitope regions, peptide 1 and peptide 2, can contact CDR loops in VH and VL of Fab.

Figure 5. Deuterium uptake plots of ETD fragment ions between TL1A alone (black) and TL1A/Fab1 (dark red). (a) ¹⁰²TVVRQTPTQHFKNQF¹¹⁶ and (b) ¹⁶⁶EIRQAGRPNKPDSIT¹⁸⁰. HDX-ETD was conducted in triplicate and the average deuterium uptake of each fragment ion was reported with standard deviation. The level of Fab1 binding-induced HDX reduction on each fragment ion was labeled in blue.

Figure 6. Residues in TL1A that are involved in mAb1 binding as determined by both HDX-ETD and computational modeling. (a) All residues are solvent exposed as shown in the TL1A monomer of its trimer crystal structure (PDB code: 2RE9). (b) A zoom-in view of the binding residues in peptide region 2 of TL1A in the TL1A/DcR3 crystal structure (PDB code: 3K51).