FIGURE 3.

Structural basis of the KVD interaction. A, structural model of Kif2C-ATP bound to tubulin. Kif2C is colored according to its three subdomains: tubulin-binding subdomain (green), P-loop subdomain (orange), and switch 1/2 subdomain (blue), defined as in kinesin-1 (17). The position of the neighboring β-tubulin (β-tubulin (2)) was modeled as in the curved assembly of two tubulins with a stathmin-like domain protein (Protein Data Bank entry 3RYC). Loop 2 of Kif2C points to and interacts with the tubulin interdimer interface. B, conformational change of Kif2C upon binding to tubulin. The tubulin-binding subdomain of Kif2C alone (Protein Data Bank entry 2HEH, shown in gray) was superimposed with the tubulin-binding subdomain of Kif2C in the structural model of Kif2C-ATP-tubulin (colored as in A), showing that the conformational change is reached through substantial rotation of P-loop subdomain (17°) and switch 1/2 subdomain (14°) of Kif2C. As a consequence, the KVD motif at the tip of loop 2 of Kif2C is well positioned to interact with the tubulin interdimer interface. C, close-up of the binding site of the Kif2C KVD motif on the tubulin interdimer interface. Color code is as in A. Black dashed lines indicate hydrogen bonds or salt bridges; red dashed lines indicate van der Waals contacts. D, SDS-PAGE analysis of the supernatant (S) and pellet (P) following ultracentrifugation of 2 μm Taxotere-stabilized microtubules after incubation with wild type Kif2C-(sN+M) or its DVK or DSK mutants. Note that the Kif2C mutants co-pellet with microtubules, indicating that their binding to microtubules is not impaired.