Side chains of Lys/Arg near transmembrane domain (TMD) membrane-water interfaces can snorkel', placing their positive charge near negatively charged phospholipid head groups; however, snorkelling's functional effects are obscure. Integrin β TMDs have such conserved basic amino acids. Here we use NMR spectroscopy to show that integrin β3 (Lys-716) helps determine β3 TMD topography. The αIIb β3 TMD structure indicates that precise β3 TMD crossing angles enable the assembly of outer and inner membrane clasps' that hold the αβ TMD together to limit transmembrane signalling. Mutation of β3 (Lys-716) caused dissociation of αIIb β3 TMDs and integrin activation. To confirm that altered topography of β3 (Lys-716) mutants activated αIIb β3, we used directed evolution of β3 (K716A) to identify substitutions restoring default state. Introduction of Pro(711) at the midpoint of β3 TMD (A711P) increased α IIb β3 TMD association and inactivated integrin α IIb β3 (A711P,K716A). β3 (Pro-711) introduced a TMD kink of 30-±-1° precisely at the border of the outer and inner membrane clasps, thereby decoupling the tilt between these segments. Thus, widely occurring snorkelling residues in TMDs can help maintain TMD topography and membrane-embedding, thereby regulating transmembrane signalling.
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