Browsing by Author "Manandhar, Pragya"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Probing the Integrin Allostery by Thermal Stability and Affinity(2017-10-12) Le, Trevor; Manandhar, PragyaαXβ2 integrin is central to the migration of myeloid cells during inflammatory response. The ligand binding domain of αXβ2, called αX I-domain, has a Mg2+-binding site (MIDAS) and an allosteric α7-helix. It has been postulated that the MIDAS site crosstalks with α7-helix and this cross-talk is associated with conformational change within the fold of the αX I-domain, yet biochemical basis of this cross-talk has yet to be elucidated. Our hypothesis is that MIDAS/α7-helix crosstalk exists in a thermodynamic equilibrium between ensembles of open and closed states. Our goal is to test how the observed conformational changes alters Mg2+-affinity to MIDAS as well as the thermal stability of the αX I-domain in solution. Conclusions. Mg2+ affinity to the intact αX-I domain is 4.848 mM ± 0.133 (Fig.X1) and the αX I-domain locked in an open-alternate conformation by I314G mutation on the αI α7-helix is 1.006 mM ± 0.050 (Fig.X2). However, the high range of melting temperature in the I314G αX I-domain suggests that the transition could potentially be bi-phasic involving two different binding-phases with the dissociation constants of 0.146 mM ± 0.020 and 3.328 mM ± 0.325. This observation is an indication that a multiple ensemble of conformations could exist between the open and closed states of the αX I-domain. These observations indicate that 1) Mg2+ stabilizes the αX-I domain and 2) smaller range of melting (40-48 ºC) of the intact αX I-domain compared to that of I314G αX I-domain (43-55 ºC) shows that the close state is less dynamic than the open state.Item Roles of Magnesium and Simvastatin in Structural Changes, Stability, and Affinity of the Ligand Binding Domain of Integrin αXβ2(2021-05) Manandhar, Pragya; Şen, Mehmet; Fox, Robert O.; Schwartz, Robert J.; Willson, Richard C.; Ma, Qing; MacKenzie, KevinMyeloid leukocytes contribute to inflammatory responses in immune dysregulations such as atherosclerosis, autoimmune encephalomyelitis, and arthritis. Leukocyte integrin, αXβ2 plays a pivotal role in their recruitment from circulation to lesions. Integrin αXβ2, known as a dendritic cell marker and complement receptor 4, is a heterodimeric cell surface receptor that bi-directionally relays cellular signals and functions in cellular trafficking, phagocytosis, and T-cell proliferation. In the regulation of these events, the ligand-binding domain of αXβ2, called the αX I-domain, acts like the gatekeeper of the bidirectional signals and relays them through affinity maturation and conformational changes from the closed to open state. A divalent cation is essential for the αX I-domain interaction with an acidic residue of a ligand. The biophysical approaches discussed here reveal that the addition of Mg2+ results in an alteration of the structure and stability of the αX I-domain in-solution. The multiplicity of the αX I-domain conformations existing in-solution is associated with its affinity regulation to physiological ligands such as fibrinogen and iC3b. The hydrophobic environment in the Metal Ion Dependent Adhesion site (MIDAS), the Mg2+-binding motif, appears to determine the ionization states of two critical MIDAS aspartates, Mg2+ creates a link from MIDAS motif to the allosteric sites, all of which contribute to the integrin activation. The local rearrangements and cation-dependent flexibility of the αX I-domain facilitate affinity maturation. These features support the rapid equilibrium of the αX I-domain between the closed and the open conformations required during rapid leukocyte activation, important for regulating leukocyte adhesiveness and migration.