Electronic and protein structural dynamics of a photosensory histidine kinase

Abstract

The bacterium Caulobacter crescentus encodes a two-component signaling protein, LovK, that contains an N-terminal photosensory LOV domain coupled to a C-terminal histidine kinase. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays regulated ATPase and autophosphorylation activity in response to visible light. Femtosecond to nanosecond visible absorption spectroscopy demonstrates congruence between full-length LovK and isolated LOV domains in the mechanism and kinetics of light-dependent cysteinyl-C4(a) adduct formation and rupture, while steady-state absorption and fluorescence line narrowing (FLN) spectroscopies reveal unique features in the electronic structure of the LovK flavin cofactor. In agreement with other sensor histidine kinases, ATP binds specifically to LovK with micromolar affinity. However, ATP binding to the histidine kinase domain of LovK has no apparent effect on global protein structure as assessed by differential Fourier transform infrared (FTIR) spectroscopy. Cysteinyl adduct formation results in only minor changes in the structure of LovK as determined by differential FTIR. This study provides insight into the structural underpinnings of LOV-mediated signal transduction in the context of a full-length histidine kinase. In particular, the data provide evidence for a model in which small changes in the tertiary/quaternary structure of LovK, as triggered by photon detection in the N-terminal LOV sensory domain, are sufficient to regulate histidine kinase activity.

Figure 1

(A) Absorption spectra of C. crescentus LovK (red) and A. sativa phot1-LOV2 (blue). (B) Cartoon illustrating light-dependent adduct formation between LovK residue C70 and C(4a) of the FMN isoalloxazine moiety. (C) Light minus dark difference absorption spectra of C. crescentus LovK (red) and A. sativa phot1-LOV2 (blue). Absorption maxima for the adduct species in LovK (390 nm) and phot1-LOV2 (397 nm) are labelled

Figure 2

Measuring LovK ATP binding affinity by fluorescence anisotropy. The dependence of signal from the fluorescent ATP analog, BODIPY-ATP, on the concentration of LovK is shown in terms of fraction of LovK bound to BODIPY-ATP. Data are fit to the Hill equation (black line; see Materials and Methods).

Figure 3

Dark recovery kinetics of LovK from the cysteinyl-C4(a) adduct state in the presence of 500 mM DMAP (black dots) and 800 mM imidazole (white dots). Biexponential fits to the data are shown as a black line (DMAP) and gray line (imidazole).

Figure 4

(A) EADS of LovK pumped at 400 nm and monitored from femtoseconds to nanoseconds. FMN singlet excited state (black line) decays to a triplet state (gray line) in 2 ns with a yield of about 70%. (B) Kinetic absorption traces at 446, 536 and 614 nm of LovK pumped at 400 nm; fit in black line.

Figure 5

Fluorescence line narrowing (FLN) spectra of LovK (upper black line) and phot1-LOV2 (lower gray line) in different spectral regions (A) 850-610 cm⁻¹, (B) 1510-1000 cm⁻¹, (C) 1650-1525 cm⁻¹ and (D) 1730-1690 cm⁻¹.

Figure 6

(A) Low temperature FTIR light minus dark spectra recorded at 150 (black), 200 (red) and 300 K (blue). (B) Comparison of light minus dark FTIR spectrum of LovK (blue line) with Phot1-LOV2 (red line) at 300K.