Structure of the ACF7 EF-Hand-GAR Module and Delineation of Microtubule Binding Determinants

Abstract

SUMMARY Spectraplakins are large molecules that cross-link F-actin and microtubules (MTs). Mutations in spectraplakins yield defective cell polarization, aberrant focal adhesion dynamics, and dystonia. We present the 2.8 Å crystal structure of the hACF7 EF1-EF2-GAR MT-binding module and delineate the GAR residues critical for MT binding. The EF1-EF2 and GAR domains are autonomous domains connected by a flexible linker. The EF1-EF2 domain is an EFβ-scaffold with two bound Ca2+ ions that straddles a N-terminal α-helix. The GAR domain has a unique α/β sandwich fold that coordinates Zn2+. While the EF1-EF2 domain is not sufficient for MT-binding, the GAR domain is and likely enhances EF1-EF2-MT engagement. Residues in a conserved basic patch, distal to the GAR domain’s Zn2+ binding site, mediate MT binding.

MACF1 Mutations Encoding Highly Conserved Zinc-Binding Residues of the GAR Domain Cause Defects in Neuronal Migration and Axon Guidance

“…The GAR domain mediates MT binding and consists of a novel zinc-binding a-b fold composed of a five-stranded anti-parallel b sheet (b1-b5) flanked by N-terminal (a1) and C-terminal (a2) a helices that pack against the b sheet to form an a/b sandwich. 3 The a1-b1 loop and the C-terminal loop flanking a2 contain four highly conserved residues (Cys7133, Cys7135, Asp7186, and Cys7188 according to Ensembl sequence MACF1-204) that coordinate the bound zinc ion. 3 Our first seven subjects (including both twins) had recurrent missense variants involving three of the four zinc-binding residues.…”

“…3 The a1-b1 loop and the C-terminal loop flanking a2 contain four highly conserved residues (Cys7133, Cys7135, Asp7186, and Cys7188 according to Ensembl sequence MACF1-204) that coordinate the bound zinc ion. 3 Our first seven subjects (including both twins) had recurrent missense variants involving three of the four zinc-binding residues. These are shown with both the standard NCBI sequence GenBank: NM_021090.5 and the longer Ensembl sequence MACF1-204 (Table S2).…”

“…Hereafter, we refer only to the MACF1-204 transcript, which has been used for most functional studies. 3…”

“…Mammalian genomes contain two spectraplakins-MACF1 (also known as ACF7) and DST (also known as BPAG1)-that function as actin-MT cross-linkers and essential integrators and modulators of cytoskeletal processes. [1][2][3] MACF1 is a large gene that expresses many isoforms, several of which are brain specific, through alternative splicing. The N terminus of the major isoforms contains two calponin-homology (CH) domains that bind actin, a plakin domain, and a long spectrin-repeat rod domain that confers flexibility.…”

“…The C terminus of all isoforms functions as a MT binding domain and contains two calcium-binding EF-hand domains, a zinc-binding GAR (growth-arrest specific 2 or Gas2-related) domain, a positively charged Gly-Ser-Arg (GSR) region, and an EB1-binding SxIP domain. [1][2][3] This work began after we recognized a complex brain malformation consisting of lissencephaly (LIS), a rare brainstem malformation with deficient midline crossing, and multiple developmental deficits including severe intellectual disability and epilepsy in three unrelated children during intergroup reviews. We next searched our large brain-malformation databases ($3,300 subjects) and identified the same pattern in another five children for a total of eight, including monozygotic twin sisters and one previously reported Japanese girl (Figures 1, 2, S1, and S2).…”