Microtubule assembly inhibitor protein consists of a rigid globule essential for its activity and highly mobile coils.
The structure of microtubule assembly inhibitor protein (MIP) was studied by proton nuclear magnetic resonance (NMR) and limited proteolysis. Spin-diffusion experiments revealed that MIP has a tightly folded structure, a "rigid globule." This globule was irreversibly denatured by heat treatment at 80 degrees C, and the denatured MIP showed little ability to inhibit microtubule assembly. This indicates that the native globular structure is essential for the activity. By spin-diffusion and spin-echo experiments, the other part of the molecule was found to be highly mobile. We termed the region "highly mobile coils." Most of the acidic residues appeared to be clustered in the highly mobile coils. By limited proteolysis using subtilisin, the mobile region was digested into shorter pieces, and a single 17-kDa fragment remained. Proton NMR spectrum of this fragment was much the same as the spin-diffusion subspectra of the rigid globule in the intact MIP molecule. Furthermore, the 17-kDa fragment was found to retain the activity to inhibit the microtubule assembly. These results indicate that MIP consists of two moieties; one domain forms a rigid globule which is essential for its activity to inhibit microtubule assembly, and the other acidic one is highly mobile and tails from the globule. The tertiary structures of these two domains appear to be independent from each other. These domains may be responsible for two different functions of MIP, the interaction with the cytoskeleton and the interaction with, for example, nuclear components.
Department of Biochemical Engineering and Science, Faculty of Computer Science, Kyushu Institute of Technology, Fukuoka, Japan.
S
S
Kotani
G
G
Kawai
H
H
Aizawa
S
S
Yokoyama
H
H
Sakai
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D003598:Cytoskeletal Proteins; D006358:Hot Temperature; D007668:Kidney; D009682:Magnetic Resonance Spectroscopy; D008870:Microtubules / Q000648:ultrastructure*; D009038:Motion; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D011489:Protein Denaturation; D011506:Proteins / Q000648:ultrastructure*; D013381:Subtilisins / Q000494:pharmacology; D013552:Swine
D000596:Amino Acids; D003598:Cytoskeletal Proteins; C057420:MIP protein, Bos taurus; D010446:Peptide Fragments; D011506:Proteins; D013381:Subtilisins