MODULATION OF MICAL ACTIVITY BY THE CALPONIN HOMOLOGY DOMAIN: STRUCTURAL, BIOCHEMICAL & THERMODYNAMIC STUDIES

MICAL;Molecule Interacting with Cas-L;CasL;Axon guidance;Monooxygenase;flavoprotein;CH;Calponin Homology;Actin-binding protein;Methionine oxidation;Biophysics

Al Qassim, SaifDevreotes, Peter N. ;

Johns Hopkins University

关键词: MICAL;    Molecule Interacting with Cas-L;    CasL;    Axon guidance;    Monooxygenase;    flavoprotein;    CH;    Calponin Homology;    Actin-binding protein;    Methionine oxidation;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37951/Acknowledgements.pdf?sequence=2&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

MICAL (Molecule Interacting with CasL) is a 1048 amino acid protein consisting of a flavin-containing monooxygenase domain (FD) with redox activity, a Calponin homology (CH) domain, a LIM (Lin-11, Isl-1, Mec-3) domain, a proline-rich region, and a C-term region containing coiled-coil ERM α-like domain. In axon guidance, MICAL is a key molecule that links the extracellular signal from semaphorins –a class of repulsive guidance cues– to the reorganization of the cytoskeleton. Axon guidance is the process by which growing axons respond to extracellular cues that guide them towards their appropriate targets. Proper axon guidance is vital in neural development processes such as neuronal cell-migration, axonal branching, path finding, and fasiculation/defasiculation. Our laboratory has previously determined the crystal structure of MICALs FD (MICALFD) and showed that it uses NADPH as the reductant. Studies showed that MICALFD and MICALFD-CH (a construct containing the FD and CH domains only) can bind and oxidize Met44 on actin filaments, thereby affecting their polymerization dynamics. CH domains are typically found in actin-binding proteins. However, modulation of MICAL activities by its non-redox CH domain is poorly understood. In this thesis work, the modulation of MICAL activity by the CH domain was characterized structurally, biochemically, and thermodynamically. The crystal structure of MICALFD-CH, determined to 2.9-Å resolution, reveals that the CH domain does not interact with the active site in the FD. Furthermore, the FD and CH domains are connected by a disordered linker, which could be important for accommodating the binding of the FD to actin. Kinetic studies reveal that the presence of the CH domain is important for substrate specificity. The structure of MICALFD-CH was used to build a possible model of the interaction of MICAL with actin. Finally, molecular dynamics simulations using this model revealed that a direct oxidation of actin’s Met44 by MICAL is possible, by an extension of the loop containing Met44 of actin. Taken together, this thesis work provides valuable insight into the means by which the CH domain of MICAL modulates its activity.