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. 2012 Apr 20;7(4):744-52.
doi: 10.1021/cb200403x. Epub 2012 Feb 6.

Structural evidence for cooperative microtubule stabilization by Taxol and the endogenous dynamics regulator MAP4

Hui Xiao  1 , Hui Wang, Xuechun Zhang, Zongcai Tu, Chloë Bulinski, Marina Khrapunovich-Baine, Ruth Hogue Angeletti, Susan Band Horwitz
Affiliations

Structural evidence for cooperative microtubule stabilization by Taxol and the endogenous dynamics regulator MAP4

Hui Xiao et al. ACS Chem Biol. .

Abstract

Microtubules (MTs) composed of αβ-tubulin heterodimers are highly dynamic polymers, whose stability can be regulated by numerous endogenous and exogenous factors. Both the antimitotic drug Taxol and microtubule-associated proteins (MAPs) stabilize this dynamicity by binding to and altering the conformation of MTs. In the current study, amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to examine the structural and dynamic properties of the MT complex with the microtubule binding domain of MAP4 (MTB-MAP4) in the presence and absence of Taxol. The changes in the HDX levels indicate that MTB-MAP4 may bind to both the outside and the luminal surfaces of the MTs and that Taxol reduces both of these interactions. The MTB-MAP4 binding induces conformational rearrangements of α- and β-tubulin that promote an overall stabilization of MTs. Paradoxically, despite Taxol's negative effects on MAP4 interactions with the MTs, its binding to the MTB-MAP4-MT complex further reduces the overall deuterium incorporation, suggesting that a more stable complex is formed in the presence of the drug.

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Figures

Figure 1
Figure 1
Global deuterium incorporation into chicken erythrocyte α- (open circle, red) and β-tubulin (filled circle, blue) in the presence of GMPCPP alone (control, solid lines), or with MAP4 added (dashed lines).
Figure 2
Figure 2
MAP4-induced alterations in deuterium incorporation referenced against GMPCPP-stabilized microtubules for (a) α-tubulin and (b) β-tubulin. Peptides resulting from the digest of the microtubule complexes with MTB-MAP4 and MTB-MAP4+Taxol are indicated in blue and red, respectively. Data denote the mean±S.D. of three separate experiments. Differences in deuteration per amino acid are expressed in mmu. Peptides are labeled with the corresponding amino acid numbers, as well as secondary structure designations based on Löwe et al. (22).
Figure 3
Figure 3
Structure of tubulin heterodimer (PDB code 1JEF): map of local HDX alterations in the presence of MAP4 (a) and MAP4+Taxol (b) referenced against GMPCPP-MTs. Peptides are color-coded as follows: yellow=insignificant change in deuterium incorporation; green=significant increase in deuterium incorporation; magenta ; red=significant reduction in deuterium incorporation with weak and strong effects and grey= undetected peptides. For clarity, only those peptides that are significantly different from those shown in a are shown in color in b. Peptides shown in grey in b represent those in which no change has occurred in deuterium incorporation plus those not detected in this experiment. Orientation of the dimer is indicated in the upper left corner: “in” refers to the inside (luminal side) of the MT, “out” to the outside, (+) to the plus end (GTP-cap) and (−) to the minus end (MT organizing center). Peptides are labeled with the corresponding amino acid numbers, as well as secondary structure designations based on Löwe et al. (22).
Figure 4
Figure 4
Interdimer interface of the tubulin dimer (PDB code 1JFF): map of local HDX alterations in the presence of MAP4 (a) and MAP4+Taxol (b). Peptides are color-coded as follows: yellow=insignificant change in deuterium incorporation; green=significant increase in deuterium incorporation; magenta and red=significant reduction in deuterium incorporation with weak and strong effects, respectively. The directional coordinates are shown in a, adjacent to each α- and β-tubulin component of the interface, with designations as indicated in Figure 3. Peptides are labeled with the corresponding amino acid numbers, as well as secondary structure designations based on Löwe et al. (22).
Figure 5
Figure 5
Intradimer interface of the tubulin dimer (PDB code 1JFF): map of local HDX alterations in the presence of MAP4 (a) and MAP4+Taxol (b). Peptides are color-coded as follows: yellow=insignificant change in deuterium incorporation; green=significant increase in deuterium incorporation; magenta and red=significant reduction in deuterium incorporation with weak and strong effects, respectively. The directional coordinates are shown in a, on top of each α- and β-tubulin component of the interface, with designations as indicated in Figure 3. Peptides are labeled with the corresponding amino acid numbers, as well as secondary structure designations based on Löwe et al. (22).
Figure 6
Figure 6
Lateral interface between adjacent protofilaments (21) : map of local HDX alterations in the presence of MAP4 (a) and MAP4+Taxol (b). The interactions between adjacent protofilaments are shown as if viewed down the length of the protofilament with the upper portion corresponding to the inside and the lower portion corresponding to the outside of the microtubule. Peptides are color-coded as follows: yellow=insignificant change in deuterium incorporation; green=significant increase in deuterium incorporation; magenta and red=significant reduction in deuterium incorporation with weak and strong effects, respectively. Secondary structure designations are based on Löwe et al. (22).
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