5 years ago

Crystal Structure of Leiomodin 2 in Complex with Actin: A Structural and Functional Reexamination

Crystal Structure of Leiomodin 2 in Complex with Actin: A Structural and Functional Reexamination
Roberto Dominguez, Zeynep Yurtsever, Malgorzata Boczkowska, Grzegorz Rebowski, Michael J. Eck


Leiomodins (Lmods) are a family of actin filament nucleators related to tropomodulins (Tmods), which are pointed end-capping proteins. Whereas Tmods have alternating tropomyosin- and actin-binding sites (TMBS1, ABS1, TMBS2, ABS2), Lmods lack TMBS2 and half of ABS1, and present a C-terminal extension containing a proline-rich domain and an actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domain that is absent in Tmods. Most of the nucleation activity of Lmods resides within a fragment encompassing ABS2 and the C-terminal extension. This fragment recruits actin monomers into a polymerization nucleus. Here, we revise a recently reported structure of this region of Lmod2 in complex with actin and provide biochemical validation for the newly revised structure. We find that instead of two actin subunits connected by a single Lmod2 polypeptide, as reported in the original structure, the P1 unit cell contains two nearly identical copies of actin monomers, each bound to Lmod2's ABS2 and WH2 domain, with no electron density connecting these two domains. Moreover, we show that the two actin molecules in the unit cell are related to each other by a local twofold noncrystallographic symmetry axis, a conformation clearly distinct from that of actin subunits in the helical filament. We further find that a proposed actin-binding site within the missing connecting region of Lmod2, termed helix h1, does not bind actin in vitro and that the electron density assigned to it in the original structure corresponds instead to a WH2 domain with opposite backbone directionality. Polymerization assays using Lmod2 mutants of helix h1 and the WH2 domain support this conclusion. Finally, we find that deleting the C-terminal extension of Lmod1 and Lmod2 results in an approximately threefold decrease in the nucleation activity, which is only partially accounted for by the lack of the WH2 domain.

Publisher URL: http://www.cell.com/biophysj/fulltext/S0006-3495(17)30804-4

DOI: 10.1016/j.bpj.2017.07.007

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