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Subdomain organization of the Acanthamoeba myosin IC tail from cryo-electron microscopy - PubMed

  • ️Thu Jan 01 2004

Comparative Study

. 2004 Aug 17;101(33):12189-94.

doi: 10.1073/pnas.0404835101. Epub 2004 Aug 9.

Affiliations

Comparative Study

Subdomain organization of the Acanthamoeba myosin IC tail from cryo-electron microscopy

Takashi Ishikawa et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Acanthamoeba myosin IC (AMIC) is a single-headed myosin comprised of one heavy chain (129 kDa) and one light chain (17 kDa). The heavy chain has head, neck (light chain-binding), and tail domains. The tail consists of four subdomains: a basic region (BR) (23 kDa) and two Gly/Pro/Ala-rich (GPA) regions, GPA1 (6 kDa) and GPA2 (15 kDa), flanking an Src homology 3 region (6 kDa). Although the AMIC head is similar in sequence, structure, and function (ATPase motor) to other myosin heads, the organization of the tail has been less clear as has its function beyond an assumed role in binding interaction partners, e.g., the BR has a membrane affinity and the GPA components bind F-actin in an ATP-independent manner. To investigate the spatial arrangement of subdomains in the tail, we have used cryo-electron microscopy and image reconstruction to compare actin filaments decorated with WT AMIC and tail-truncated mutants of various lengths. The BR forms an oval-shaped feature, approximately 40 A long, that diverges obliquely from the head, extending azimuthally around the actin filament and toward its barbed end. GPA2 and GPA1 are located together on the inner (actin-proximal) side of the tail, close enough to act in concert in binding the same or another actin filament. The outer face of the BR is strategically exposed for membrane or vesicle binding.

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Figures

Fig. 1.
Fig. 1.

Subdomain organization and actin filament-binding properties of full-length AMIC and truncated variants. (A) Schematic diagram of the domain compositions of the AMIC mutants analyzed in this study. The domain boundaries are from ref. . (B) SDS/PAGE of the purified proteins (reproduced from ref. 13). HC, heavy chain; LC, light chain. (C) Typical cryo-electron micrographs and diffraction patterns of AMIC-decorated actin filaments. A few key layer-lines are indexed.

Fig. 2.
Fig. 2.

Surface renderings of the 3D structures of actin filaments decorated with various AMIC-associated constructs. (A) T1. (B) T4. (C) T5. (D) T6. (E) WT. (F) ΔSH3. Note the very thin connection (arrow) between the head–neck and tail (arrowhead) in the T4 construct (B) and its progressive thickening in the longer tail constructs (C–F, arrows). The two bars denote the difference in length between the T1 and T4 heads. A side-by-side comparison of T1 and WT is given in Fig. 6, which is published as

supporting information

on the PNAS web site.

Fig. 3.
Fig. 3.

Difference densities mapped on surface renderings and in horizontal sections. (A) The difference between T4 and T1 (red) is overlaid on the T1 reconstruction (light blue). The density of actin (34) is shown in green. (B)T5–T4 overlaid on T4. (C)WT–T6 overlaid on T6. In B and C, extra density that we interpret as being GPA1-associated is marked by arrows in B: enhanced density on the other side of BR (arrowheads) is interpreted as an ordering effect.

Fig. 4.
Fig. 4.

Docking of the Dictyostelium myosin IE head (32) into the cryo-EM density map of actin filaments decorated with WT-AMIC. The atomic model of actin is shown in green, and the myosin IE head domain is shown in yellow. The yellow and blue lines illustrate the difference between the rigor actin-binding aspects of the heads of AMIC and myosin II.

Fig. 5.
Fig. 5.

Schematic representation of the relative positions of the various subdomains of AMIC as bound to F-actin in the rigor conformation. The GPA regions are positioned where they might readily engage the same filament (on a conformational change in the neck or on release of the head) or another parallel and adjacent actin filament. The BR, facing outward, is appropriately placed to allow ready access to membranes.

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