Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner.
Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function.
From the Institute of Biochemistry and Biophysics (IQUIFIB), School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina, Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany, burgardt@qb.ffyb.uba.ar.
Noelia Inés
NI
Burgardt
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.
Andreas
A
Schmidt
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.
Annika
A
Manns
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.
Alexandra
A
Schutkowski
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.
Günther
G
Jahreis
Graduate Institute of Natural Products, Center of Excellence for Environmental Medicine, Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 807, Taiwan, and.
Yi-Jan
YJ
Lin
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.
Bianca
B
Schulze
Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Department of Enzymology, 06099 Halle (Saale), Germany.
Antonia
A
Masch
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany.
Christian
C
Lücke
Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle (Saale), Germany, Martin-Luther-University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Department of Enzymology, 06099 Halle (Saale), Germany.
Matthias
M
Weiwad
D020816:Amino Acid Motifs; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058105:Polymerization; D011485:Protein Binding; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D002147:Calmodulin; D008869:Microtubule-Associated Proteins; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D014404:Tubulin; C000606846:PIN1 protein, human; C000606848:PIN4 protein, human; D019696:Peptidylprolyl Isomerase; D002118:Calcium
calcium; calmodulin (CaM); chemical shift perturbation (CSP) analysis; microtubule-associated protein (MAP); nuclear magnetic resonance (NMR); parvulin; protein conformation; protein-protein interaction; tubulin