Dual effects of transmembrane proline residues on integrin function

2015-06-17With 50% of all drug targets, membrane proteins present an important subcategory for structural characterization, even though they code for less than a third of all proteins in the human genome. Due to specific properties, the structure of membrane proteins are not easily characterized by conventional methods, therefore NMR presents a unique tool for that purpose. Integrins are a type I heterodimeric receptors that are essential for cell adhesion and migration. Integrins are large heterodimeric membrane receptors that tie the extracellular matrix to the cytoskeleton (Harburger and Calderwood, 2009). It is essential to both the arrest of bleeding at sites of vascular injury and pathological thrombosis culminating in heart attack and stroke. Integrin bi‐directional transmembrane (TM) signaling involves the dissociation of a complex formed by the α–β TM segments, which is accompanied by large rearrangements of its extracellular domains. ❧ TM segment sequences of the 8 β human subunits are well conserved, especially within a distinct positive charged residue at the inner membrane clasp, β₃(K716). In our research, we have shown that charge variation of the lipid system influences K716’s amide-lipid interaction at the C-terminal membrane interface, therefore defining a regulation mechanism for αIIb β₃ heterodimer formation. Site-directed mutation of K716 caused integrin to achieve its high-affinity state by stabilizing its monomeric species. Using directed evolution of β₃(K716A), we identified a substitution, A711P, to restore the integrin α IIb β₃ default low-affinity state. Quantitative dimerization analysis, utilizing ITC, NMR and FRET, independently verified an increase in TM domain dimer formation upon proline mutation, therefore recovering the low-affinity state. Structural analysis of the monomeric β₃(A711P/K716A) identified a kink of 30 ± 1° at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Subsequently, the integrin αIIb β₃(A711P) heterodimer revealed a strong glycine packing at the OMC compared to the wt, hence its complex represents the first structure of a heterodimeric TM receptor of its kind and reveals a dimerization interface of captivating complexity. ❧ Utilizing NMR and MD simulation, we determined proline's extended role at the N-terminal membrane-water interface of single-pass α-helical TM domains in integrin αIIb and interleukin βc. The structural characterization of these mutants revealed proline's function as N-terminal helix-cap and linker terminator. Therefore, αIIb (P965) establishes an equilibrium between the length of the TM helix and its N-terminal linker domain, regulating signal transmission towards the extracellular domains. These results describe signaling and activation tendencies of members in the integrin α family. Taken together, the structural knowledge presented in this thesis provides insights to the disease mechanism and will form a scientific basis for future therapeutic treatments of stroke and cancer