Bent to Bind: Exploiting the Programmed Cell Death-1 (PD-1) Receptor Plasticity to Design Pembrolizumab H3 Loop Mimics

Checkpoint blockade of the Programmed cell Death-1 (PD-1) immunoreceptor with its ligand 1 (PD-L1) by the monoclonal antibody pembrolizumab provided compelling clinical results among various cancer types, yet the molecular mechanism by which this drug blocks the PD-1:PD-L1 binding interface and reactivates exhausted T cells remains unclear. To address this question, we examined the conformational motion of PD-1 associated with the binding of pembrolizumab. The largely overlooked innate plasticity of both PD-1 C’D and FG loops appears crucial to closing in the receptor edges on the drug. Herein, we describe how PD-1 bends to initiate the formation of a deep binding groove (371 ų) across several epitopes while engaging pembrolizumab. Our analysis ultimately provided a rational design for mimicking the pembrolizumab H3 loop [RDYRFDMGFD] as a PD-1 inhibitor. A series of H3 loop mimics were synthesized and their folding characterized by CD and NMR spectroscopy. As a result, a first-in-class <i>b</i>-hairpin peptide inhibitor of the PD-1/PD-L1 interface was identified (IC₅₀ of 0.6 ± 0.2 μM). Overall, this study demonstrates that the dynamic groove formed between the C’D and FG loops of PD-1 is an attractive target for the development of peptide-based PD-1 inhibitors.

Bent to Bind: Exploiting the Programmed Cell Death-1 (PD-1) Receptor Plasticity to Design Pembrolizumab H3 Loop Mimics

Checkpoint blockade of the Programmed cell Death-1 (PD-1) immunoreceptor with its ligand 1 (PD-L1) by the monoclonal antibody pembrolizumab provided compelling clinical results among various cancer types, yet the molecular mechanism by which this drug blocks the PD-1:PD-L1 binding interface and reactivates exhausted T cells remains unclear. To address this question, we examined the conformational motion of PD-1 associated with the binding of pembrolizumab. The largely overlooked innate plasticity of both PD-1 C’D and FG loops appears crucial to closing in the receptor edges on the drug. Herein, we describe how PD-1 bends to initiate the formation of a deep binding groove (371 ų) across several epitopes while engaging pembrolizumab. Our analysis ultimately provided a rational design for mimicking the pembrolizumab H3 loop [RDYRFDMGFD] as a PD-1 inhibitor. A series of H3 loop mimics were synthesized and their folding characterized by CD and NMR spectroscopy. As a result, a first-in-class <i>b</i>-hairpin peptide inhibitor of the PD-1/PD-L1 interface was identified (IC₅₀ of 0.6 ± 0.2 μM). Overall, this study demonstrates that the dynamic groove formed between the C’D and FG loops of PD-1 is an attractive target for the development of peptide-based PD-1 inhibitors.

Conformational rearrangements in the N-domain of Escherichia coli FepA during ferric enterobactin transport

The Escherichia coli outer membrane receptor FepA transports ferric enterobactin (FeEnt) by an energy- and TonB-dependent, but otherwise a mechanistically undetermined process involving its internal 150-residue N-terminal globular domain (N-domain). We genetically introduced pairs of Cys residues in different regions of the FepA tertiary structure, with the potential to form disulfide bonds. These included Cys pairs on adjacent β-strands of the N-domain (intra-N) and Cys pairs that bridged the external surface of the N-domain to the interior of the C-terminal transmembrane β-barrel (inter-N–C). We characterized FeEnt uptake by these mutants with siderophore nutrition tests, [59Fe]Ent binding and uptake experiments, and fluorescence decoy sensor assays. The three methods consistently showed that the intra-N disulfide bonds, which restrict conformational motion within the N-domain, prevented FeEnt uptake, whereas most inter-N–C disulfide bonds did not prevent FeEnt uptake. These outcomes indicate that conformational rearrangements must occur in the N terminus of FepA during FeEnt transport. They also argue against disengagement of the N-domain out of the channel as a rigid body and suggest instead that it remains within the transmembrane pore as FeEnt enters the periplasm.

Emission enhancement of GFP chromophore in aggregated state via combination of self-restricted effect and supramolecular host–guest complexation

The emission response of GFP chromophore in aggregated state is greatly enhanced more than 100-fold due to the inhibition of conformational motion and the reduction of strong π–π interaction.

Emission enhancement and application of synthetic green fluorescent protein chromophore analogs

Emission enhancement and application of GFPc analogs are achieved via chemical modification or physical encapsulation due to conformational motion inhibition.

Ion pair-induced conformational motion in calix[4]arene-strapped calix[4]pyrroles

Cone- and conformationally mobile calix[4]arene-strapped calix[4]pyrroles bind cesium salts via various different binding modes.