In Silico Investigations of the Metastable States of αX I-domain and Characterization of its First Novel Competitive Inhibitor

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Integrins, particularly leukocyte integrins, play a pivotal role in immune responses. The immunomodulatory heterodimer transmembrane glycoprotein αXβ2, also known as Complement Receptor 4 (CR4, CD11c/CD18) and one of the four β2 leukocyte integrins, is predominantly expressed in human myeloid cell types like dendritic cells and macrophages. Dysregulation of this receptor is implicated in hyperproliferative and inflammatory diseases. Understanding the dynamic nature of αXβ2 is crucial for designing targeted therapeutics for autoimmune disorders and malignancies while avoiding global immunodeficiency risks. Our three-pronged investigation utilized diverse methodologies to dissect the activation mechanisms, antagonist interactions, and structural dynamics of αX, alongside comparison studies with αM and αL I-domains. Firstly, employing constant-pH hybrid nonequilibrium molecular dynamics-monte carlo (CPHMD-MC) simulations, we delved into the pH and cation impact on the αX I-domain. Our results unveiled pH-dependent conformational changes and emphasized the crucial role of Mg2+, shedding light on the thermodynamic equilibrium and activation mechanism of αX. Expanding our scope to the αXβ2 integrin, we explored its interaction dynamics with the competitive antagonist, simvastatin. Through simvastatin-αX I-domain complex simulations, we elucidated the dynamics and residue interaction network, aligning with Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR) spectra. Uncovering interactions within the Metal Ion-Dependent Adhesion Site (MIDAS), our findings laid the groundwork for understanding the antagonistic activity of simvastatin, opening avenues for drug development. The final study focused on unbiased and biased metadynamics simulations to unravel the structural dynamics of αX, αM, and αL I-domains. Identifying metastable states as LA-l and IA-l, we gained crucial insights into their activation mechanisms. Integrating Nuclear Magnetic Resonance (NMR) bias impacted the FEL shape, enhancing the robustness of our findings through comparative analysis with prior studies. In summary, our comprehensive investigation advanced the understanding of leukocyte integrins, offering a foundation for targeted therapeutic interventions. The insights into metastable states, antagonist interactions, and structural dynamics provide a holistic perspective on integrin biology, guiding future experimental validations and drug discovery efforts.

Leukocyte integrins, αXβ2, Complement Receptor 4, constant-pH hybrid nonequilibrium molecular dynamics-monte carlo, simvastatin, competitive antagonist, free energy landscape, meta dynamics metainference, metastable states, NMR-biased, conformational dynamics
Portions of this document appear in: Manandhar, Pragya, Zahra Mazhar, Omar Abousaway, Collins Aboagye, Zeinab Moussa, Daniel Lim, Tannon Yu, James Byrnes, James M. Briggs, and Mehmet Sen. "Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2." Cell reports 40, no. 8 (2022).