Article

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: 3.01). 01/2005; 43(51):16184-92. DOI: 10.1021/bi048092i
Source: PubMed

ABSTRACT 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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    • "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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    • "Since the A9a helix also interacts with the Ja helix (Fig. 4D, top, white circles), the modification at His-495 may alter the interaction of the A9a helix with the Ja helix. This altered interaction could lead to partial unfolding and displacement of the Ja helix away from the LOV2 core in the mutant (Fig. 4D, bottom right, green arrows), as has been observed in the mutated oat phot1 (Harper et al., 2004). Moreover, this displacement could cause the observed instability of the mutant phot1. "
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    • "While the FMN molecule is noncovalently associated with the LOV domain in darkness, upon absorption of BL, a reversible photocycle is initiated such that the activated FMN forms a covalent adduct with a nearby Cys residue in the LOV domain (Christie et al., 1999, 2002; Salomon et al., 2000). Although their photocycles are similar, the LOV1 domain is thought primarily to regulate receptor di/multimerization (Salomon et al., 2004; Nakasako et al., 2008; Nakasone et al., 2013), whereas LOV2 appears to regulate the C-terminal PKD of phots through a novel BL-induced derepression mechanism (Christie et al., 2002; Harper et al., 2003, 2004; Jones et al., 2007; Jones and Christie, 2008; Nakasako et al., 2008; Tokutomi et al., 2008). In the absence of light, the LOV2 domain is folded in a way that causes steric inhibition of the PKD (Figure 2A). "
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