Abstract
The vertebrate-specific proteins astrotactin-1 and 2 (ASTN-1 and ASTN-2) are integral membrane perforin-like proteins known to play critical roles in neurodevelopment, while ASTN-2 has been linked to the planar cell polarity pathway in hair cells. Genetic variations associated with them are linked to a variety of neurodevelopmental disorders and other neurological pathologies, including an advanced onset of Alzheimer's disease. Here we present the structure of the majority endosomal region of ASTN-2, showing it to consist of a unique combination of polypeptide folds: a perforin-like domain, a minimal epidermal growth factor-like module, a unique form of fibronectin type III domain and an annexin-like domain. The perforin-like domain differs from that of other members of the membrane attack complex-perforin (MACPF) protein family in ways that suggest ASTN-2 does not form pores. Structural and biophysical data show that ASTN-2 (but not ASTN-1) binds inositol triphosphates, suggesting a mechanism for membrane recognition or secondary messenger regulation of its activity. The annexin-like domain is closest in fold to repeat three of human annexin V and similarly binds calcium, and yet shares no sequence homology with it. Overall, our structure provides the first atomic-resolution description of a MACPF protein involved in development, while highlighting distinctive features of ASTN-2 responsible for its activity.
Highlights
Perforin-like proteins (PLPs) have been identified in all forms of cellular life except, currently, Archaebacteria [1]
We verified that the expressed protein had remained intact such that epidermal growth factor (EGF) repeats 2 and 3 (EGF-2 and EGF-3) were present in the crystallized protein, they were not resolved in the structure, presumably due to disorder, though small-angle X-ray scattering (SAXS) data indicate that the domains are folded
Other integral membrane membrane attack complex-perforin (MACPF)/CDC proteins include perforin-2, a macrophage-specific form of perforin involved in bacterial killing [33,58], and torso-like, which plays a role in early development of the Drosophila embryo [43,68]
Summary
Perforin-like proteins (PLPs) have been identified in all forms of cellular life except, currently, Archaebacteria [1]. They represent a sub-branch of the largest known family of pore-forming proteins, the membrane attack complexperforin/cholesterol-dependent cytolysin (MACPF/CDC) family [2,3]. First as part of the complement membrane attack complex (MAC) [8,9] and in the form of perforin-1 [10,11], which delivers granzymes from cytotoxic cells into target antigen presenting cells. The only basis for MACPF/CDC activity properly established so far is the oligomerization of many subunits in order to generate a pore-forming complex [15,16,17,18,19]. It has been argued both that oligomerization to a complete ring of subunits is required for pore formation [7,22], and that incomplete rings (arcs) of MACPF/
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