Independent functions for the N- and C-termini in the overlap region of tropomyosin

Tropomyosin (TM) is a coiled-coil that binds head-to-tail along the helical actin filament. The ends of 284-residue tropomyosins are believed to overl ap by about nine amino acids. The present study investigates the function o f the N- and C-terminal overlap regions. Recombinant tropomyosins were prod uced in Escherichia coli in which nine amino acids were truncated from the N-terminal, C-terminal, or both ends of striated muscle alpha-tropomyosin ( TM9a) and TM2 (TM9d), a nonmuscle alpha-tropomyosin expressed in many cells . The two isoforms are identical except for the C-terminal 27 amino acids e ncoded by exon 9a (striated) or exon 9d (TM2). Removal of either end greatl y reduces the actin affinity of both tropomyosins in all conditions and the cooperativity with which myosin promotes tropomyosin binding to actin in t he open state. N-Terminal truncations generally are more deleterious than C -terminal truncations. With TM9d, truncation of the N-terminus is as delete rious as both for myosin S1-induced binding. None of the TM9d variants bind s well to actin with troponin (+/-Ca2+). TM9a with the truncated N-terminus binds more weakly to actin with troponin (-Ca2+) than when the C-terminus is removed but more strongly than when both ends are removed; the actin bin ding of all three forms is cooperative. The results show that the ends of T M9a, though important, are not required for cooperative function and sugges t they have independent functions beyond formation of an overlap complex. T he nonadditivity of the TM9d truncations suggests that the ends may primari ly function as a complex in this isoform. A surprising result is that all v ariants bound with the same affinity, and noncooperatively, to actin satura ted with myosin S1. Evidently, end-to-end interactions are not required for high-affinity binding to acto-myosin S1.