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Figure 3.
Figure 3. Structure of the Second TOG Domains from Mini
Spindles and Stu2p
Ribbon diagram of the TOG2 domain from
Msps (A) and Stu2p (B) with the six HEAT-like repeats
represented in shades of similar color and labeled A–F. The
conserved and nonconserved regions (faces A and B, respectively)
are indicated. (C) Least-squares fit of Msps (color) and Stu2p
(gray) with TOG2 domains shown in cylindrical helix
representation. (D) Individual TOG2 HEAT-like repeats are shown
for Msps and Stu2p in ribbons format in similar orientations
after global least-squares fit of each TOG2 domain. The
definitive α helix kink that defines HEAT repeats is evident in
the α2 helices of Msps HEAT-like repeats C and D and Stu2p
HEAT-like repeats C and F. (E) 90° rotations of the Msps
TOG2 domain about its long axis shown at left in ribbons for
orientation and at right in CPK representation for conservation
mapping. TOG2 residues with 80% identity across species are
represented in green, 80% conservation in yellow (see Figure
S2). (E) 2F[o] − F[c] electron density map at 1.7 Å
resolution of the Stu2p TOG2 structure contoured at 1.0 σ
showing the surface exposed and highly conserved KEKK loop of
HEAT-like repeat C. (G) 2F[o] − F[c] electron density map at
2.1 Å resolution of the Msps TOG2 structure contoured at
1.0 σ showing the surface exposed W292 residue and the buried
R295-D331 salt bridge. Inset (upper left) indicates the relative
orientation of the TOG domain (F and G). (H) Gel filtration
tubulin binding assays for wild-type (WT) and mutant Msps
TOG1-2. Single or double mutations of the conserved TOG domain
tryptophan (TOG1: W21E, TOG2: W292E) are indicated above the
chromatogram. Tubulin alone, black; Msps TOG1-2 WT alone, red.
The plot indicates absorption at 280 nm on the y axis (mAU) and
elution volume in ml along the x axis.
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