Structural Insights into Modulation of Mical by its Calponin Homology (CH) Domain

Abstract

MICAL (Molecule Interacting with CasL) is a 1048 amino acid protein consisting of a monooxygenase domain (FD) with redox activity, a Calponin homology domain (CH), a LIM 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. Proper axon guidance, the process by which growing axons respond to extracellular cues that guide them towards their appropriate targets, 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 domain (MICALFD) and showed that it uses NADPH as the reductant. Studies showed that MICALFD and MICALFD-CH can bind and oxidize Met44 on actin filaments, thereby affecting their polymerization dynamics. However, modulation of these MICAL activities by its non-redox domains is poorly understood. To structurally characterize the modulation by the CH domain, we determined the crystal structure of MICALFD-CH to 3.0-A resolution. The structure reveals that the CH domain does not interact with the active site in the FD domain. Furthermore, the FD and CH domains are flexible with respect to each other; MICALFD-CH crystallized in two different crystal forms, and no electron density was observed for the 18-residue linker between the two domains. In actin-binding proteins with tandem CH domains, the flexibility of the domains with respect to each other is important for binding F-actin. Similarly, the flexibility of the two domains in MICALFD-CH may be important in optimizing the binding to F-actin such that Met44 is more accessible to the active site.