Sedimentation velocity and crystal structure of She2p. (A) Diagram showing the domain structure of Myo4p and its association with She3p. The motor domain contains the actin and ATP-binding sites followed by the lever arm, which binds six light chains/CaM. The rod of Myo4p binds to She3p. The C terminus of the Myo4p heavy chain forms a globular tail domain. (B) Crystal structure of She2p (Protein Data Bank accession no. 1XLY). The two chains of the dimer are shown in green and cyan. The location of the two point mutants, S120Y and L130Y, are shown in space-filling representation. S120Y is at the interface of the crystallographic dimer, whereas L130Y is at the proposed interface for tetramer formation (Müller et al., 2009). The four differences between WT and WT* are shown in yellow space-filling representation. The Cys to Ser mutations were made to prevent formation of higher oligomers of She2p (see C). A single α helix that protrudes from the side of She2p (red) is identified as all or part of the binding site for She3p. The mRNA-binding site is indicated in tan. (C) WT She2p exhibited a polydisperse profile when analyzed by sedimentation velocity in the analytical ultracentrifuge. In contrast, when four Cys residues were mutated to Ser (C14S/C68S/C106S/C180S; yellow spheres in B), the protein showed a single homogeneous peak that sedimented at 5.4 S. This construct, called WT*, was used as the backbone for further mutants described in this paper. Protein concentration was 8 µM She2p tetramer. A representative centrifuge run is illustrated (n = 3). OD, optical density.