Disruption of the LOV−Jα Helix Interaction Activates Phototropin Kinase Activity †

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
Biochemistry (Impact Factor: 3.02). 01/2005; 43(51):16184-92. DOI: 10.1021/bi048092i
Source: PubMed


Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

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    • "m being fully understood in all three photoreceptor classes , although some key results emerge to shed light onto the principal mechanisms : More than 10 years ago , ground - breaking results from NMR spectroscopy demonstrated a reordering or even an unfolding of the helix ( named J α ) bridging a LOV2 domain and its signal - transduction module ( Harper et al . , 2004a , b ; Herman et al . , 2013 ) , and thus , displayed a first molecular concept for signal propagation . Subsequent experiments with other LOV domains yielded evidence that also dimerization reactions can occur after light excitation ( Buttani et al . , 2007 ; Möglich and Moffat , 2007 ; Nakasako et al . , 2008 ; Zayner et al . , 2012 ; "
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    Frontiers in Bioscience 10/2015; 2. DOI:10.3389/fmolb.2015.00049 · 3.52 Impact Factor
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    • "ght - oxygen - voltage domains undergo versatile light dependent interactions . In the best - studied LOV domain , LOV2 from Avena sativa phototropin , light - induced thioeither bond formation between a cysteine residue and the FMN chromophore leads to partial unfolding of the C - terminal α - helix ( named Jα ) from the rest of the LOV2 domain ( Harper et al . , 2004 ) . This conformation change has been widely used to construct light - controllable proteins in allosteric or steric manners ( Lee et al . , 2008 ; Strickland et al . , 2008 ; Moglich et al . , 2009 ; Wu et al . , 2009 ; Ohlendorf et al . , 2012 ) . Wu et al . ( 2009 ) constructed photoactivatable small GTPase Rac1 ( PA - Rac1 ; Figure "
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    Frontiers in Molecular Neuroscience 08/2015; 8:37. DOI:10.3389/fnmol.2015.00037 · 4.08 Impact Factor
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    • "Ja-helix displacement therefore represents an integral component of the phototropin light switch. Indeed, artificial disruption of the Ja-helix from the LOV2 core through targeted mutagenesis uncouples this mode of regulation and leads to phot1 activation in the absence of light (Harper et al. 2004, Jones et al. 2007, Kaiserli et al. 2009). "
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    Plant and Cell Physiology 12/2014; 56(3). DOI:10.1093/pcp/pcu196 · 4.93 Impact Factor
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