Unusual Ca(2+)-calmodulin binding interactions of the microtubule-associated protein F-STOP.
F-STOP is a microtubule-associated protein that stabilizes microtubules in a calmodulin (CaM)-dependent manner. All members of the stable tubule only polypeptide (STOP) family have a central domain that contains nearly identical multiple repeats, and a CaM binding motif is present in multiple copies within this domain. We present here an analysis of this CaM binding interaction and find that it is highly unusual in nature. For this work, we synthesized two model peptides of a single STOP central repeat motif and analyzed their binding to CaM by fluorescence, circular dichroism, infrared and NMR spectroscopy. Both peptides bind to CaM with an affinity of 4 microM, similar to that of the native protein. Results indicate that the peptides bind CaM in an atypical manner. Binding is highly dependent on the concentration of cations, indicating that it is to some extent electrostatic. Further, IR and CD analysis shows that, in contrast to typical CaM binding reactions, CaM does not change in helical structure on binding. NMR mapping confirms that CaM remains in extended conformation on binding a single STOP peptide. Binding of a single peptide to CaM occurs principally in the CaM C-terminal region, and the C-terminal domain of CaM effectively competes for STOP binding. Our results establish that CaM binds STOP in an unusual manner, involving mainly the C-terminus of CaM, thus leaving CaM potentially accessible for another binding partner at the N-terminus. This intriguing possibility could be of physiological importance in F-STOP mediated CaM regulation of microtubule dynamics or stability, specifically during mitosis where CaM and STOP colocalize.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002942:Circular Dichroism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D013050:Spectrometry, Fluorescence / Q000379:methods; D014268:Trifluoperazine / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D008869:Microtubule-Associated Proteins; D014268:Trifluoperazine; D014364:Tryptophan; D002118:Calcium