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. 2016 Oct 4;24(10):1719-1728.
doi: 10.1016/j.str.2016.06.026. Epub 2016 Sep 8.

Structure and Dynamics of PD-L1 and an Ultra-High-Affinity PD-1 Receptor Mutant

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Free PMC article

Structure and Dynamics of PD-L1 and an Ultra-High-Affinity PD-1 Receptor Mutant

Roberta Pascolutti et al. Structure. .
Free PMC article

Abstract

The immune checkpoint receptor PD-1 and its ligand, PD-L1, have emerged as key regulators of anti-tumor immunity in humans. Recently, we reported an ultra-high-affinity PD-1 mutant, termed high-affinity consensus (HAC) PD-1, which shows superior therapeutic efficacy in mice compared with antibodies. However, the molecular details underlying the action of this agent remain incompletely understood, and a molecular view of PD-1/PD-L1 interactions in general is only beginning to emerge. Here, we report the structure of HAC PD-1 in complex with PD-L1, showing that it binds PD-L1 using a unique set of polar interactions. Biophysical studies and long-timescale molecular dynamics experiments reveal the mechanisms by which ten point mutations confer a 35,000-fold enhancement in binding affinity, and offer atomic-scale views of the role of conformational dynamics in PD-1/PD-L1 interactions. Finally, we show that the HAC PD-1 exhibits pH-dependent affinity, with pseudo-irreversible binding in a low pH setting akin to the tumor microenvironment.

Figures

Figure 1
Figure 1
HAC PD-1 and PD-L1 structure. (A) The overall structure of HAC PD-1 in complex with PD-L1 shows two copies of each molecule in each asymmetric unit, with domain swapping in PD-L1. HAC PD-1 is depicted in purple and blue, while PD-L1 is represented in green and yellow. The complex is shown in ribbon presentation. (B) SEC-MALS analysis shows that the two molecules interact in a 1:1 complex. (C) Superimposition of wild-type PD-1 apo (3RRQ), wild-type PD-1 (4ZQK) and HAC PD-1 (blue) shows slight differences in conformations. (D) Wild-type PD-1 (PDB ID: 4ZQK) and HAC PD-1 are depicted in cyan and blue respectively. Superimposition of HAC PD-1 with wild-type PD-1 shows a major conformational change in the loop containing Pro72.
Figure 2
Figure 2
ITC measurements of wild-type PD-1 or HAC PD-1 binding to PD-L1. The values for ΔH and ΔS were obtained by fitting a single binding site model to the ITC data and are shown with standard error values. The top panel shows examples of raw data indicating the titration of wild-type PD-1 (left) or HAC PD-1 (right) into an isothermal calorimetry cell containing PD-L1. The bottom panels represent plots of heat released during the isothermal reaction vs. molar ratio for the interaction of wild-type PD-1 (left) or HAC PD-1 (right) with PD-L1. The values for ΔG and ΔS in HAC/PD-L1 interaction should be interpreted with some caution due to the sharp slope of the curve caused by high binding affinity.
Figure 3
Figure 3
Close-up views of HAC PD-1 mutations at the interface with PD-L1. Within the complex structure, wild-type PD-1 and PD-L1 (PDB: 4ZQK) are represented in cyan and yellow respectively (A and C). HAC PD-1 and PD-L1 are depicted in blue and yellow respectively (B and D). The complexes are shown in ribbon presentation. Mutation M70E leads to the formation of new hydrogen bonds (B) compared to the wild-type structure (A), stabilizing the interface of interaction with PD-L1. A similar behavior is shown by the mutations Y68H and K78T in HAC-PD-1 (D) compared to the wild-type protein (C).
Figure 4
Figure 4
M70E seems to be the main factor to improve stability. (A) Five most populated conformations of HAC PD-1 and wild-type PD-1 shown respectively in red and grey. (B) Five most populated conformations of HAC PD-1 when bound to PD-L1 (blue) and HAC PD-1 free (red). (C) Five most populated conformations of wild-type PD-1/PD-L1 and wild-type PD-1 free depicted respectively in cyan and grey. (D) Temporal evaluations of the distances of M70 Sδ - R139 Cζ in wild-type PD-1 and E70 Cδ - R139 Cζ in HAC PD-1.
Figure 5
Figure 5
Y68H and K78T mutations contribute to stability, but at lower extent. (A) Temporal evolution of distances between the side chains of residue 68 on PD-1 and the Cγ of D122 on PD-L1. For HAC PD-1/PD-L1, Nε on H68 (PD-1) and the Cγ on D122 (PD-L1) have been used to calculate the distances. For the wild-type PD-1/PD-L1 system, side chain Oη on Y68 (PD-1) and Cγ on D122 (PD-L1) were used. (B) Temporal evolution of distances between the side chains of residue 78 on PD-1 and the Cγ of D122 on PD-L1. For the HAC PD-1/PD-L1 system, Oγ on T78 (PD-1) and the Cγ on D122 (PD-L1) were considered to calculate the distances. For the wild-type PD-1/PD-L1 system, Cδ on K78 (PD-1) and Cγ on D122 (PD-L1) were studied. (C) Temporal evolution of distances between the side chains of residue 70 on PD-1 and the Cζ of R125 on PD-L1. For HAC PD-1/PD-L1, Cδ on E70 (PD-1) and Cζ of R125 (PD-L1) were used to calculate the distances. For the wild-type PD-1/PD-L1 system, side chain Sδ on M70 (PD-1) and Cζ of R125 (PD-L1) were used. (A–C) In all the panels, HAC PD-1/PD-L1 is represented in black and wild-type PD-1/PD-L1 in red. (D) Graphical representation of the interactions undertaken by E70 in HAC PD-1/PD-L1. HAC PD-1 is depicted in blue, while PD-L1 is represented in yellow. The proteins are shown in ribbon presentation.
Figure 6
Figure 6
pH-dependent interaction between HAC PD-1 and PD-L1. (A–D) Representative surface plasmon resonance (SPR) sensorgrams of HAC PD-1 binding to immobilized PD-L1. (E–F) KD values extrapolated from SPR analysis at different pH are represented with their s.e. For the experiment carried out at pH 5.5, the KD was not measurable (n.m.)

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