The AtMAP65-1 cross-bridge between microtubules is formed by one dimer.
The microtubule-associated protein AtMAP65-1 from Arabidopsis thaliana dimerizes and forms 25 nm cross-bridges between microtubules, but the exact mechanism is unknown. Here, we used the predicted three-dimensional structure of AtMAP65-1 as a basis for analyzing the actual cross-bridging in detail. Fold-recognition predicts that AtMAP65-1 contains four coiled-coil domains and a flexible extended loop. The length of these coiled-coil domains is about 25 nm, suggesting that one molecule could span the gap, hence forming an antiparallel overlapping dimer instead of an end-to-end dimer. We then tested this model by using truncations of AtMAP65-1. EDC {[3-(dimethylamino) propyl] carbodiimide} cross-linking analysis indicated that the N-terminus of the rod domain of AtMAP65-1 (amino acids 1-339) binds to the C-terminus of the rod domain (amino acids 340-494) and also participates in connecting the two antiparallel proteins in the cross-bridge. Nevertheless, microtubules can still form bundles in the presence of AtMAP65-1 340-587 (amino acids 340-587) or AtMAP65-1 1-494 (amino acids 1-494). Comparing the cold stability of microtubule bundles induced by full-length AtMAP65-1 with that of AtMAP65-1 340-587 or AtMAP65-1 1-494, we conclude that AtMAP65-1 495-587 acts as a flexible extended loop, playing a crucial role in binding to and stabilizing microtubules in the AtMAP65-1 cross-bridge.
Li
Hua
H
State Key Laboratory of Plant Physiology and Biochemistry, China Agricultural University, Beijing 100094, PR China.
Mao
Tonglin
T
Zhang
Ziding
Z
Yuan
Ming
M
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D018506:Gene Expression Regulation, Plant; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation
D029681:Arabidopsis Proteins; C492901:MAP65-1 protein, Arabidopsis; D008869:Microtubule-Associated Proteins