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A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

Zhang D, Asenjo AB, Greenbaum M, Xie L, Sharp DJ, Sosa H - PLoS ONE (2013)

Bottom Line: To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A.Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells.The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York, United States of America ; State Key Lab of Reproductive Medicine, College of Basic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China.

ABSTRACT
Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

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Kinesin HD electrostatic surface potentials in tubulin binding areas.(A) Ribbon representation of the KLP10AHD-tubulin-MT spiral complex model (PDB IC: 3J2U [10]) showing the two tubulin binding sites at opposite sides of the HD. The Kin-Tub-1 area corresponds to the putative MT binding site, common to all kinesins, but in this case it mediates binding to a curved tubulin protofilament. The Kin-Tub-2 area mediates binding of the kinesin-13-HD-curved-tubulin complex to the MT and the formation of spirals wrapping the MT. Mutating kinesin-13 class conserved positively charged residues in the Kin-Tub-2 area (KLP10A residues K306, K350, and K399 highlighted as blue atom spheres) disrupt these interactions and prevents the spirals from wrapping around MTs [13]. The location of the kinesin-13 Loop-2 (L2) is indicated. (B) Electrostatic surface potential comparison of Kin-Tub-1 and Kin-Tub-2 areas of a kinesin-1 (HsKIF5B) and a kinesin-13 (DmKLP10A). Color scale inset: -10 (red) to +10 (blue) kcal/mol·e. (C) Kin-Tub-2 area of several kinesin families. The corresponding kinesin family is indicated below each HD structure. (D) Kin-Tub-2 area of several kinesin-13s (all within the Kinesin-13B/MCAK subfamily [14]). In B-D the name corresponding to each kinesin sequence are indicated with an italics two letter prefix corresponding to the organism origin (Ce: Caenorhabditis elegans; Cg: Cricetulus griseus; Dr: Danio rerio; Ds: Drosophila melanogaster; Hs: Homo Sapiens; Mm: Mus musculus; Xl: Xenopus laevis) and the PDB IC below (references [46–52]. When no atomic structures were available, a homology based model (HBM) was calculated using the program Modeller [53].
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pone-0073075-g001: Kinesin HD electrostatic surface potentials in tubulin binding areas.(A) Ribbon representation of the KLP10AHD-tubulin-MT spiral complex model (PDB IC: 3J2U [10]) showing the two tubulin binding sites at opposite sides of the HD. The Kin-Tub-1 area corresponds to the putative MT binding site, common to all kinesins, but in this case it mediates binding to a curved tubulin protofilament. The Kin-Tub-2 area mediates binding of the kinesin-13-HD-curved-tubulin complex to the MT and the formation of spirals wrapping the MT. Mutating kinesin-13 class conserved positively charged residues in the Kin-Tub-2 area (KLP10A residues K306, K350, and K399 highlighted as blue atom spheres) disrupt these interactions and prevents the spirals from wrapping around MTs [13]. The location of the kinesin-13 Loop-2 (L2) is indicated. (B) Electrostatic surface potential comparison of Kin-Tub-1 and Kin-Tub-2 areas of a kinesin-1 (HsKIF5B) and a kinesin-13 (DmKLP10A). Color scale inset: -10 (red) to +10 (blue) kcal/mol·e. (C) Kin-Tub-2 area of several kinesin families. The corresponding kinesin family is indicated below each HD structure. (D) Kin-Tub-2 area of several kinesin-13s (all within the Kinesin-13B/MCAK subfamily [14]). In B-D the name corresponding to each kinesin sequence are indicated with an italics two letter prefix corresponding to the organism origin (Ce: Caenorhabditis elegans; Cg: Cricetulus griseus; Dr: Danio rerio; Ds: Drosophila melanogaster; Hs: Homo Sapiens; Mm: Mus musculus; Xl: Xenopus laevis) and the PDB IC below (references [46–52]. When no atomic structures were available, a homology based model (HBM) was calculated using the program Modeller [53].

Mentions: In previous work we found that kinesin-13s can form oligomeric rings and spirals around MTs, mediated by an additional tubulin binding site on the HD that we called Kin-Tub-2 [13] (Figure 1A). A comparison of the electrostatic surface potential of the HD of several kinesins (Figure 1B–D) reveals that Kin-Tub-2 area of kinesin-13s is different from other kinesins. In most kinesins, this area is negatively charged, but in kinesins-13s, particularly in the 13B.MCAK/KIF2 subfamily [14], it tends to be positively charged. Some noted exceptions to this generalization are human KIF2B and D. melanogaster KLP59D, which are more electronegative in the Kin-Tub-2 site. It would be of interest to investigate whether this distinction is related to the common mitotic roles that these two kinesins play in human [6] and D. melanogaster cells [15].


A second tubulin binding site on the kinesin-13 motor head domain is important during mitosis.

Zhang D, Asenjo AB, Greenbaum M, Xie L, Sharp DJ, Sosa H - PLoS ONE (2013)

Kinesin HD electrostatic surface potentials in tubulin binding areas.(A) Ribbon representation of the KLP10AHD-tubulin-MT spiral complex model (PDB IC: 3J2U [10]) showing the two tubulin binding sites at opposite sides of the HD. The Kin-Tub-1 area corresponds to the putative MT binding site, common to all kinesins, but in this case it mediates binding to a curved tubulin protofilament. The Kin-Tub-2 area mediates binding of the kinesin-13-HD-curved-tubulin complex to the MT and the formation of spirals wrapping the MT. Mutating kinesin-13 class conserved positively charged residues in the Kin-Tub-2 area (KLP10A residues K306, K350, and K399 highlighted as blue atom spheres) disrupt these interactions and prevents the spirals from wrapping around MTs [13]. The location of the kinesin-13 Loop-2 (L2) is indicated. (B) Electrostatic surface potential comparison of Kin-Tub-1 and Kin-Tub-2 areas of a kinesin-1 (HsKIF5B) and a kinesin-13 (DmKLP10A). Color scale inset: -10 (red) to +10 (blue) kcal/mol·e. (C) Kin-Tub-2 area of several kinesin families. The corresponding kinesin family is indicated below each HD structure. (D) Kin-Tub-2 area of several kinesin-13s (all within the Kinesin-13B/MCAK subfamily [14]). In B-D the name corresponding to each kinesin sequence are indicated with an italics two letter prefix corresponding to the organism origin (Ce: Caenorhabditis elegans; Cg: Cricetulus griseus; Dr: Danio rerio; Ds: Drosophila melanogaster; Hs: Homo Sapiens; Mm: Mus musculus; Xl: Xenopus laevis) and the PDB IC below (references [46–52]. When no atomic structures were available, a homology based model (HBM) was calculated using the program Modeller [53].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755979&req=5

pone-0073075-g001: Kinesin HD electrostatic surface potentials in tubulin binding areas.(A) Ribbon representation of the KLP10AHD-tubulin-MT spiral complex model (PDB IC: 3J2U [10]) showing the two tubulin binding sites at opposite sides of the HD. The Kin-Tub-1 area corresponds to the putative MT binding site, common to all kinesins, but in this case it mediates binding to a curved tubulin protofilament. The Kin-Tub-2 area mediates binding of the kinesin-13-HD-curved-tubulin complex to the MT and the formation of spirals wrapping the MT. Mutating kinesin-13 class conserved positively charged residues in the Kin-Tub-2 area (KLP10A residues K306, K350, and K399 highlighted as blue atom spheres) disrupt these interactions and prevents the spirals from wrapping around MTs [13]. The location of the kinesin-13 Loop-2 (L2) is indicated. (B) Electrostatic surface potential comparison of Kin-Tub-1 and Kin-Tub-2 areas of a kinesin-1 (HsKIF5B) and a kinesin-13 (DmKLP10A). Color scale inset: -10 (red) to +10 (blue) kcal/mol·e. (C) Kin-Tub-2 area of several kinesin families. The corresponding kinesin family is indicated below each HD structure. (D) Kin-Tub-2 area of several kinesin-13s (all within the Kinesin-13B/MCAK subfamily [14]). In B-D the name corresponding to each kinesin sequence are indicated with an italics two letter prefix corresponding to the organism origin (Ce: Caenorhabditis elegans; Cg: Cricetulus griseus; Dr: Danio rerio; Ds: Drosophila melanogaster; Hs: Homo Sapiens; Mm: Mus musculus; Xl: Xenopus laevis) and the PDB IC below (references [46–52]. When no atomic structures were available, a homology based model (HBM) was calculated using the program Modeller [53].
Mentions: In previous work we found that kinesin-13s can form oligomeric rings and spirals around MTs, mediated by an additional tubulin binding site on the HD that we called Kin-Tub-2 [13] (Figure 1A). A comparison of the electrostatic surface potential of the HD of several kinesins (Figure 1B–D) reveals that Kin-Tub-2 area of kinesin-13s is different from other kinesins. In most kinesins, this area is negatively charged, but in kinesins-13s, particularly in the 13B.MCAK/KIF2 subfamily [14], it tends to be positively charged. Some noted exceptions to this generalization are human KIF2B and D. melanogaster KLP59D, which are more electronegative in the Kin-Tub-2 site. It would be of interest to investigate whether this distinction is related to the common mitotic roles that these two kinesins play in human [6] and D. melanogaster cells [15].

Bottom Line: To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A.Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells.The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York, United States of America ; State Key Lab of Reproductive Medicine, College of Basic Medicine, Nanjing Medical University, Nanjing, Jiangsu, China.

ABSTRACT
Kinesin-13s are microtubule (MT) depolymerases different from most other kinesins that move along MTs. Like other kinesins, they have a motor or head domain (HD) containing a tubulin and an ATP binding site. Interestingly, kinesin-13s have an additional binding site (Kin-Tub-2) on the opposite side of the HD that contains several family conserved positively charged residues. The role of this site in kinesin-13 function is not clear. To address this issue, we investigated the in-vitro and in-vivo effects of mutating Kin-Tub-2 family conserved residues on the Drosophila melanogaster kinesin-13, KLP10A. We show that the Kin-Tub-2 site enhances tubulin cross-linking and MT bundling properties of KLP10A in-vitro. Disruption of the Kin-Tub-2 site, despite not having a deleterious effect on MT depolymerization, results in abnormal mitotic spindles and lagging chromosomes during mitosis in Drosophila S2 cells. The results suggest that the additional Kin-Tub-2 tubulin biding site plays a direct MT attachment role in-vivo.

Show MeSH
Related in: MedlinePlus