De Paula, V. S. | bioRxiv Science

bioRxiv2024

Regulating IL-2 immune signaling function via a core allosteric structural network

Human interleukin-2 (IL-2) is a crucial cytokine for T cell regulation, with therapeutic potential in cancer and autoimmune diseases. However, IL-2s pleiotropic effects across different immune cell types often lead to toxicity and limited efficacy. Previous efforts to enhance IL-2s therapeutic profile have focused on modifying its receptor binding sites. Yet, the underlying dynamics and intramolecular networks contributing to IL-2 receptor recognition remain unexplored. This study presents a detailed characterization of IL-2 dynamics compared to two engineered IL-2 mutants, "superkines" S15 and S1, which exhibit biased signaling towards effector T cells. Using NMR spectroscopy and molecular dynamics simulations, we demonstrate significant variations in core dynamic pathways and conformational exchange rates across these three IL-2 variants. We identify distinct allosteric networks and excited state conformations in the superkines, despite their structural similarity to wild-type IL-2. Furthermore, we rationally design a mutation (L56A) in the S1 superkines core network, which partially reverts its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. Our results reveal that IL-2 superkine core dynamics play a critical role in their enhanced receptor binding and function, suggesting that modulating IL-2 dynamics and core allostery represents an untapped approach for designing immunotherapies with improved immune cell selectivity profiles. HighlightsO_LINMR and molecular dynamics simulations revealed distinct conformational dynamics and allosteric networks in computationally re-designed IL-2 superkines compared to wild-type IL-2, despite their similar crystal structures. C_LIO_LIThe superkines S1 and S15 exhibit altered sampling of excited state conformations at an intermediate timescale, with slower conformational exchange rates compared to wild-type IL-2. C_LIO_LIA rationally designed mutation (L56A) in the S1 superkines core allosteric network partially reverted its dynamics, receptor binding affinity, and T cell signaling behavior towards that of wild-type IL-2. C_LIO_LIOur study demonstrates that IL-2 core dynamics play a critical role in receptor binding and signaling function, providing a foundation for engineering more selective IL-2-based immunotherapies. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/617024v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1e298adorg.highwire.dtl.DTLVardef@195b602org.highwire.dtl.DTLVardef@1e06205org.highwire.dtl.DTLVardef@4351e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

A slow-exchange conformational switch regulates off-target cleavage by high-fidelity Cas9

The Cas9 endonuclease is broadly used for genome engineering applications by programming its single-guide RNA, where high specificity is required. Although mechanistic understanding of DNA cleavage in the CRISPR-Cas9 system has been guided by crystallographic structures and single-molecule FRET experiments, the role of conformational plasticity within its DNA recognition lobe (REC) for target accuracy remains elusive. Through NMR analysis of milliseconds-timescale dynamics, we show that the REC3 domain exchanges between a major, closed and a minor, open conformation. We find that a single mutation in the HiFi Cas9 variant (R691A) increases conformational dynamics in REC3, eliciting a global transition to the open conformation which is regulated by an intramolecular salt-bridge network. These observations suggest a mechanism for reduced off-target recognition by switching on a global conformational exchange process to allow REC3 scanning for proper base pairing. Our data establish a framework for rational engineering Cas9 variants with improved target discrimination.

biochemistry↗

bioRxiv2019

Interleukin-2 druggability is modulated by global conformational transitions controlled by a helical capping switch

Interleukin-2 (IL-2) is a small -helical cytokine that regulates immune cell homeostasis through its recruitment to a high-affinity heterotrimeric receptor complex (IL-2R/IL-2R{beta}/{gamma}c). IL-2 has been shown to have therapeutic efficacy for immune diseases by preferentially expanding distinct T-cell compartments, and several Treg-biasing anti-IL-2 antibodies have been developed for combination therapies. The conformational plasticity of IL-2 plays an important role in its biological actions by modulating the strength of receptor and drug interactions. Through a NMR analysis of milliseconds-timescale dynamics of free mIL-2 we identify a global transition to an auto-inhibitory conformation controlled by an -helical capping \"switch\". Binding to either an anti-mouse IL-2 mAb or a small molecule inhibitor near the AB loop induces changes in dynamics at the core of the structure, and perturbations of the capping hydrogen-bond network abrogate Ab binding by destabilizing the auto-inhibitory conformation. Our results highlight a paradigm for designing precision therapeutics targeting a continuum of IL-2 conformational states.

biochemistry↗