15LOV-Domain Structure, Dynamics, and Diversity

Sean Crosson

15.1Overview

Light, oxygen, or voltage (LOV) domains, a subset of the PER-ARNT-SIM (PAS) su-perfamily (Taylor and Zhulin, 1999), were originally identified as the loci for blue-light absorption in the plant photoreceptor kinases known as phototropins (Christieet al., 1999). These domains have since been shown to act as photosensory modulesin DNA-binding and F-box proteins that regulate circadian rhythms in fungi (Chenget al., 2003) and higher plants (Imaizumi et al., 2003), and have been identified in thegenomes of a variety of other organisms including several species of photosyntheticand non-photosynthetic eubacteria (Crosson et al., 2003; Losi et al., 2002). With theexception of the phototropins, which possess two tandem LOV domains adjacent toa carboxy-terminal serine/threonine kinase (Huala et al., 1997), these photosensorymodules are typically found as a single copy at the amino-terminal region of a struc-turally and functionally diverse set of proteins (Crosson et al., 2003). LOV domainscontain approximately 110 amino acids, bind a single molecule of f lavin, and under-go a unique photochemical transformation in response to blue-light absorption inwhich a conserved cysteine residue forms a covalent bond with the 4a carbon of thef lavin cofactor (Crosson and Moffat, 2002; Salomon et al., 2001). In the phototropins,light-driven formation of this cysteinyl-C(4a) adduct leads to upregulation of the car-boxy-terminal serine/threonine kinase (Briggs and Christie, 2002). The function ofLOV domains in proteins other than phototropin is not as well understood.

The molecular and structural basis of how LOV domains regulate the activity of thekinase domain of phototropins, or indeed any other domain to which they are at-tached, is an active area of investigation. This chapter focuses on recent work de-scribing the structure and dynamics of LOV domains in their dark and photoexcitedstates. Included is a comparative analysis of LOV-domain structure that discusses therelation to other PAS domains and the structurally similar GAF domain family (Ar-avind and Ponting, 1997). In addition, an updated analysis of proteins containingLOV domains outlining the diversity in their domain structure and taxonomic distri-bution is included.

Handbook of Photosensory Receptors. Edited by W. R. Briggs, J. L. SpudichCopyright © 2005 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN 3-527-31019-3

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15.2LOV Domain Architecture and Chromophore Environment

X-ray crystallographic studies of Chlamydomonas phototropin LOV1 (Fedorov et al.,2003) and maidenhair fern phy3 LOV2 (Crosson and Moffat, 2001) reveal that LOVdomains exhibit a prototypical PAS fold (Pellequer et al., 1998) consisting of a five-stranded antiparallel β-sheet f lanked by the helix-turn-helix motif αA/αB, a single 310

helical turn α’A, and the 15-residue connector helix αC (Figure 15.1 A). A least-squares fit of the refined coordinates of Chlamydomonas phototropin LOV1 and fernphy3 LOV2 confirms that the secondary structural elements and tertiary architectureof LOV1 and LOV2 are nearly identical (main chain root-mean-square devia-tion = 0.73 Å). Embedded in the PAS fold is a single molecule of f lavin mononu-cleotide (FMN) that, in the dark state, is non-covalently bound to the core of the pro-tein by a series of polar interactions with the pyrimidine moiety and nonpolar inter-actions with the dimethylbenzene moiety of the isoalloxazine ring. Additional hydro-gen-bond and charge-charge interactions stabilize the ribityl side chain and terminalphosphate within the LOV fold (Figure 15.1 B). To date, all LOV domains have beenshown to bind FMN with the exception of the LOV domains of the Neurospora circa-dian regulators, White Collar 1 (Wc-1) and VIVID (VVD). Wc-1 binds f lavin adeninedinucleotide (FAD) and requires this cofactor for its function (He et al., 2002), whileVVD can bind both FAD and FMN when expressed in E. coli. No structural informa-

15 LOV-Domain Structure, Dynamics, and Diversity

αC

βA βB

N

C

αA

αB

α'AβC

βD

βEN

SH

OH

N

N

NH

N

O

O

O

OH

OH

P OO

O

N

N

N

N

N

N

O

O

N

O

N

NH

H

C57/966 Q120/1029

N89/998

O

N99/1008

Q61/970

V103/1012

N56/965

D31/940

R58/967

R74/983

NH

O

O

NH

O

A B

C57/966

Figure 15.1 LOV domain architecture and co-factor-binding environment. (A) Ribbon dia-gram of maidenhair fern phy3 LOV2 (PDB ac-cession number 1G28). Secondary structureelements are marked on the diagram; nomen-clature follows reference (Crosson and Moffat,2001). The conserved cysteine side chain isshown attached to the amino terminal end ofhelix α’A and is labeled with a circle and an ar-row. The FMN cofactor is shown in the core of

the domain. (B) Hydrogen bond network be-tween the LOV domain polypeptide and theFMN cofactor (dashed lines). The 1.9 Å struc-ture of Chlamydomonas phototropin LOV1(PDB accession number 19NL) was used todetermine hydrogen bonds. Residues formingbonds are labeled with the first number corre-sponding to the Chlamydomonas phototropinLOV1 structure and the second correspondingto the phy3 LOV2.

325

tion is available on how the LOV domain accommodates the adenine nucleotide moi-ety of FAD, which is bound to the ribityl phosphate and is thus predicted to be posi-tioned outside of the f lavin-binding core of the LOV fold.

A conserved cysteine residue, corresponding to C966 in fern phy3 LOV2 and C57in Chlamydomonas phototropin LOV1, f lanks the si face of the f lavin isoalloxazinering. This residue is in nearly identical positions in the LOV1 and LOV2 crystal struc-tures (Figure 15.2). In the 2.7 Å dark-state structure of phy3 LOV2 solved at roomtemperature (Crosson and Moffat, 2001), the Sγ of C966 is 4.2 Å from C(4a). Thus,facile rotation about the Cα–Cα bond combined with small movements in either theprotein main chain or in the f lavin cofactor is sufficient to bring this conserved cys-teine within covalent bonding distance of C(4a). The 1.9 Å dark-state structure ofChlamydomonas phot LOV1 solved at cryogenic temperatures (Fedorov et al., 2003)reveals two conformers of the cysteine side chain. The predominant conformer (at70 ± 10% occupancy) is similar to the refined position of C966 in phy3 LOV2. The sec-ond conformer is rotated ∼80º about the Cα–Cβ bond, bringing the Sγ within 3.5 Å ofC(4a) (Figure 15.2). Evidence for two conformers of this conserved cysteine in thedark-state structure of phy3 LOV2 is present in electron density maps calculated froma partially refined structure determined at cryogenic temperatures (S. Crosson andK. Moffat, unpublished results). It is uncertain what role temperature has on the for-mation of this minor conformer and whether this structure is important for LOV do-main photochemistry and function under biological conditions. Regardless, the con-served cysteine is closely positioned to f lavin atom C(4a) in the crystal structures ofLOV1 and LOV2, allowing for adduct formation without a large conformationalchange in the protein.

15.3Photoexcited-State Structural Dynamics of LOV Domains

While X-ray crystallographic and NMR structural analysis of proteins can provide agreat deal of insight into function, traditional versions of these techniques provideonly a static, time-averaged structure with little or no information on molecular mo-tion. Understanding small- and large-scale protein motions is necessary to decipherprocesses ranging from enzyme-substrate specificity to allostery. LOV domains pro-vide an excellent model system to understand the dynamics of protein structure be-cause they are relatively small and exhibit a self-contained photocycle (Salomon et al.,2000). Thus illumination with blue light can initiate structural changes in the proteinthat can be probed using both steady-state and time-resolved crystallography andNMR. Recently, several investigators have taken advantage of this property of LOVdomains and solved steady-state photoexcited structures of phototropin LOV1 andLOV2 domains in both crystals and solution. These experiments are beginning toprovide a picture of LOV-domain signaling with downstream partners.

Changes in LOV domain structure upon blue light illumination were first docu-mented in a set of one-dimensional 13C, 15N, and 31P NMR experiments using oatphototropin LOV2 containing an isotopically-enriched FMN chromophore (Salomon

15.2 LOV Domain Architecture and Chromophore Environment

326

et al., 2001). These experiments demonstrated that the photoproduct was indeed acysteinyl-C(4a) adduct and revealed changes in the chemical environments of thepolypeptide backbone, the terminal phosphate, and the ribityl chain of FMN in re-sponse to photon absorption. The three-dimensional structure of a LOV domain in aphotoexcited state was later solved by collecting X-ray diffraction data on a single crys-tal of fern phy3 LOV2 under continuous illumination at room temperature (Crossonand Moffat, 2002). While this steady state structure allowed the direct observation ofa covalent bond between the conserved cysteine (C966) and the FMN C(4a) carbon,there were surprisingly very few changes in the overall structure of the protein as ev-idenced by difference Fourier maps calculated against the dark-state structure factoramplitudes (Crosson and Moffat, 2001) (Figure 15.3 A). These data demonstrate that,in the context of the 104 amino acids visible in the phy3 LOV2 electron density maps,protein motion in response to photon absorption is small and is concentrated aroundthe isoalloxazine ring of the FMN cofactor. The largest motion between the dark- andilluminated-state structures is in the conserved cysteine side chain, which undergoesa simple chi1 rotation to bring the Sγ sulfur within an appropriate distance to form

15 LOV-Domain Structure, Dynamics, and Diversity

C966-Cα

C966-Cβ

C966-Sγ

C57-Cα

C57-Cβ

C57-Sγ(∼70% occupancy) C57-Sγ

C(4a)

Figure 15.2 Dark-state conformers of the con-served cysteine side chain in LOV1 and LOV2.The dark-state structure of phy3 LOV2 (1G28)reveals one conformer of the conserved cys-teine (labeled C966 with bonds colored red).The dark-state structure of Chlamydomonasphototropin LOV1 (19NL) has two conformersof the cysteine side chain (labeled C57 with

bonds colored blue and green); the predomi-nant conformer in LOV1 (at ∼70%) is in a sim-ilar position to the cysteine side chain positionin the dark-state structure of phy3 LOV2. The4a carbon of the isoalloxazine ring of FMN islabeled. Atoms are colored by elements: car-bon, green; nitrogen, blue; oxygen, red; phos-phorus, pink; sulfur, yellow.

327

the covalent adduct with C(4a). Additionally, the f lavin ring rotates ∼8° so thatresidues with hydrogen bonds to the isoalloxazine moiety can maintain these inter-actions (Figure 15.3 B). Similar studies conducted on the light state of the Chlamy-domonas phototropin LOV1 domain at cryogenic temperatures yielded similar results(Fedorov et al., 2003). Namely, electron density for the covalent cysteinyl-C(4a) adductis clearly present with protein structural changes confined to the areas around thef lavin ring. However, this illuminated-state structure of LOV1 shows slightly differ-ent geometry at the conserved cysteine (C57) as well as some small movement in theregion of the terminal phosphate of FMN that is not evident in phy3 LOV2 structure.

While these crystallographic experiments provide valuable information on thestructure of the cysteinyl adduct state, the limited light-induced conformationalchanges seen in these structures fail to explain how adduct formation can signalthrough the surrounding LOV domain to result in kinase activation. A possible mod-el for this process has been provided by recent solution NMR experiments on oat pho-totropin LOV2 (Harper et al., 2003). These multidimensional NMR experiments werecarried out on a construct of LOV2 containing an additional 40 amino acids after thelast C-terminal residues evident in the crystal structure of phy3 LOV2. This C-termi-nal extension is conserved in all phototropin LOV2 domains, including phy3 LOV2.Intriguingly, roughly 20 of these residues form an amphipathic α-helix that docksagainst the five-stranded antiparallel β-sheet of the core LOV fold (Figure 15.4 A).Pulsed illumination of this LOV2 construct causes dramatic changes in 15N/1HHSQC and other spectra, indicating extensive blue light-induced structural changes.Identical experiments conducted on mutant LOV2 protein lacking the conserved cys-

15.3 Photoexcited-State Structural Dynamics of LOV Domains

αC

α'A

αC

α'A

A B

βC

βD

βE

βC

βD

βE

Figure 15.3 Variable ligand binding propertiesof the PAS domain family. A least squares su-perposition of the structural coordinates offern phy3 LOV2 (Crosson and Moffat, 2001),Bradyrhizobium japonicum FixL-PAS (Gong etal., 1998), Halorhodospira halophila PYP (Borg-stahl et al., 1995),and Klebsiella pneumoniaeCitA-PAS (Reinelt et al., 2003) shows the lig-and that binds each of these PAS domains tooccupy a similar region of the structure.(A) Colored circles (FMN, yellow; heme, red;para-hydroxycinnamic, green; citrate, purple)

are drawn onto the polypeptide structure ofphy3 LOV2. (B) Ball-and-stick diagram usingthe same color scheme shows, in greater de-tail, how FMN, heme, para-hydroxycinnamicacid, and citrate are positioned relative to eachother. Secondary structure elements aremarked with arrows (beta sheet) and circles(helices). Panel B represents the LOV foldrotated counter-clockwise 45° across the planeof the page with respect to the orientation ofphy3 LOV2 in panel A.

328

teine reveal no spectral changes in the lit state, suggesting that changes in the wild-type LOV2 domain are a direct result of cysteinyl-C(4a) adduct formation.

A comparison of a variety of parameters, including NMR chemical shifts, 2H ex-change protection factors, as well as increased susceptibility to proteolysis, demon-strates that while the core LOV fold is slightly destabilized in the adduct state, it re-tains the same overall secondary and tertiary structure. However, the C-terminal am-phipathic helix, which is not present in the LOV2 crystal structures, is displaced fromthe β-sheet and unfolds in response to illumination (Figure 15.4 B). This adduct-in-duced change in secondary and tertiary structure likely serves as an allosteric switchcontrolling the activity of the C-terminal kinase domain, as mutations in the con-served cysteine which prevent the light-induced structural changes also render ki-nase activity completely insensitive to illumination. Helix undocking and unfoldingfrom the β-sheet of the PAS/LOV fold also occurs in the photoexcited state of PYP(Hoff et al., 1999). Indeed, protein-protein interactions through the β-sheet may be ageneral feature of several PAS-mediated signaling processes (Erbel et al., 2003). Ad-ditional experiments on mutant LOV domains and larger LOV constructs are neces-sary to understand how adduct formation results in helix undocking from the β-sheetand how this undocking subsequently regulates the activity of the C-terminal kinase.

15.4Comparative Structural Analysis of LOV Domains

As mentioned earlier, LOV domains form a subset of the PAS structural superfami-ly. A previous comparative structural analysis of phy3 LOV2 to the PAS domains ofBradyrhizobium japonicum FixL (Gong et al., 1998), Halorhodospira halophila photoac-tive yellow protein (PYP) (Borgstahl et al., 1995), and the human ERG potassiumchannel (Cabral et al., 1998) revealed a high degree of structural homology eventhough sequence homology between these domains is low (Crosson and Moffat,2001). Lack of sequence homology between the PAS domains has created difficultiesin annotation of these ubiquitous protein modules. Indeed, two of the five PAS struc-tures deposited in the Protein Data Bank (the periplasmic domain of Klebsiella pneu-moniae CitA (Reinelt et al., 2003) and the N-terminal domain of the human ERGpotassium channel (Cabral et al., 1998)) were not known to be PAS domains until theywere solved. This combination of sequence plasticity and conservation of structuresuggests that the PAS fold can be utilized in many different ways.

An example of how PAS architecture has been adapted for different functions is ev-ident in the variable ligand-binding properties of PAS domains. Many PAS proteinsfunction as sensors that relay cellular signals in direct response to ligand bindingand/or physicochemical changes at the ligand-binding site. Among the small molec-ular ligands that have been shown to bind PAS domains are: 1) iron protoporphyrinIX in the rhizobial oxygen-sensor kinase, FixL (Gilles-Gonzalez et al., 1991); 2) para-hydroxycinnamic acid in the bacterial photosensor, PYP (Baca et al., 1994; Hoff et al.,1994); 3) f lavin adenine dinucleotide (FAD) in the aerotaxis sensor of E. coli (Bibikovet al., 2000), the NifL redox sensor of Azotobacter vinelandii (Hill et al., 1996), and the

15 LOV-Domain Structure, Dynamics, and Diversity

32915.4 Comparative Structural Analysis of LOV Domains

~8∞ ring tiltFMN

C966

310 helix-α'A

I943

G1027

S930

Q1029 N1008

N998

FMN

C966 F1010

A

B

Figure 15.4 Conformational change in theFMN binding pocket of photoexcited LOV2.(A) Fourfold noncrystallographic symmetry-av-eraged light-minus-dark difference Fouriermap contoured at ±4σ in which σ is the root-mean-square value of the electron density. Theconserved cysteine and the f lavin ring for thedark (blue) and the photoexcited (yellow)structures are shown. Negative difference den-sity (blue) and positive density (yellow) indi-cate Cys and ring motion upon illumination.(B) Side chains exhibiting significant displace-ments between the dark (blue) and photoex-cited (yellow) structures in response to cys-teinyl-f lavin C(4a) adduct formation. Hydro-gen bonds between the protein and FMN co-factor in the dark and photoexcited structures

are indicated by blue and yellow dotted lines,respectively. A 2.6 to 3.5 Å range for hydrogenbonding was used. Atoms are colored by ele-ments: nitrogen, light blue; oxygen, red; sulfur,green. Atoms colored blue in the dark struc-ture and yellow in the photoexcited structureare carbon. In addition to the residues exhibit-ing motion in phy3 LOV2, the cryo-illuminatedstructure of Chlamydomonas phototropinLOV1 (Fedorov et al., 2003) shows additionaldisplacements in Asn56, which forms a hydro-gen bond with the ribityl chain, in the phos-phate of FMN, and in Arg58, which forms asalt bridge with the phosphate. Figure fromreference (Crosson and Moffat, 2002); reprint-ed with permission of the American Society ofPlant Biologists.

330

photosensory LOV domain of Neurospora Wc-1 (He et al., 2002); 4) f lavin mononu-cleotide (FMN) in the photosensory LOV domains of Arabidopsis and other pho-totropins, ZTL, FKF1, and LKP2 (Imaizumi et al., 2003); 5) small aromatic com-pounds in mammalian PAS kinase (Amezcua et al., 2002); and 6) citrate in the CitAreceptor histidine kinase of Klebsiella (Reinelt et al., 2003). Thus, PAS domains areable to bind a chemically and structurally distinct range of ligands and communicateinformation from the ligand binding site to intra- and intermolecular partners. A dis-cussion of the structural basis of this phenomenon in LOV domains will follow in alater section.

A least-squares comparison of the four types of ligand-binding PAS domain struc-tures available in the Protein Data Bank, including PYP, the LOV domain of pho-totropins, the FixL heme-binding domain, and the periplasmic citrate-bindingdomain of CitA, shows that all exhibit high structural homology at the level of the pro-tein main chain (less than 3.0 Å root mean square deviation). The most structurallyconserved region of all PAS structures is the five-stranded antiparallel β-sheet. Thiselement of secondary/tertiary structure exhibits remarkable conservation, with a rootmean square deviation of approximately 1 Å between structures [see Figure 15.3 A of(Crosson and Moffat, 2001)]. All four of the ligand-binding PAS domains positiontheir ligands in a similar region within the core of the fold suggesting a commonmode of communication from the ligand-binding core to the surface of the protein(Figure 15.5).

Bioinformatic analysis of sequence from the phytochrome family of red-light pho-toreceptors has identified additional domains that are homologous to PAS/LOV do-mains. Namely, the bilin-binding GAF and PHY domains of this versatile photore-ceptor family, which are positioned between several predicted PAS domains in thephytochrome polypeptide, are predicted to exhibit a PAS-like fold (Montgomery andLagarias, 2002). Indeed, crystallographic analysis of a yeast GAF protein confirms thestructural similarity between PAS and GAF domains (Ho et al., 2000). Thus phyto-chome is likely constructed of multiple repeating units of a structurally similar fold(Montgomery and Lagarias, 2002). How these PAS/GAF/PHY structural units cometogether to form the tertiary/quaternary structure of the holophytochrome dimer isnot known. Nevertheless, it is notable that both the phototropin and phytochromefamilies of plant photoreceptors utilize very similar structural modules for signaling.

15.5LOV-Domain Diversity

Genetic, biochemical, and biophysical studies have revealed a great deal about thestructure and function of LOV domains from the phototropin family. Notably, aBLAST search of GenBank using a LOV f lavin-binding consensus sequence(Crosson and Moffat, 2001) reveals numerous non-phototropin LOV proteins inplants, fungi and eubacteria that are predicted to bind f lavin and exhibit a canonicalLOV photocycle (Crosson et al., 2003). While f lavin binding and photochemistry hasonly been confirmed in a handful of these proteins (Imaizumi et al., 2003; Losi et al.,

15 LOV-Domain Structure, Dynamics, and Diversity

331

2002; Salomon et al., 2000), conservation of the f lavin-binding consensus is a strongpredictor that they contain bona f ide LOV domains. Besides the ZTL (Somers et al.,2000), FKF1 (Nelson et al., 2000) and LKP2 (Schultz et al., 2001) circadian regulators

15.5 LOV-Domain Diversity

Jα/αD

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αA

αB

αC

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N

C

A

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N

N

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N

O

O

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N

N

NH

N

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S

ConservedCysteine

hνdark

Core PAS/LOV fold10x increase in globalhydrogen exchange

1H, 15N and 13Cchemical shift changes

C-terminal helix of LOVundocks and unfolds

B

Figure 15.5 Light induced tertiary structurechanges in oat phototropin LOV2. (A) Ribbondiagram of oat phototropin1 LOV2 solved bymultidimensional NMR (structural coordi-nates courtesy of the Gardner Lab, Universityof Texas Southwestern Medical Center at Dal-las). Diagram shows the C-terminal helical ex-tension (Jα/αD) that is not present in thecrystal structures of phy3 LOV2 and pho-totropin LOV1. This amphipathic helix docksagainst the β-sheet of the PAS/LOV fold. The

flavin cofactor and conserved cysteine areshown in the core of the fold. (B) Cartoonsummary of oat phototropin1 LOV2 NMR da-ta shows C-terminal helix undocking and un-folding, an increase in global hydrogen/deu-terium exchange in the core PAS/LOV fold,widespread changes in 1H, 13C, and 15N chem-ical shifts, and increased proteolysis in re-sponse to adduct formation. Panel B adaptedfrom Figure S3 of reference (Harper et al.,2003).

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of Arabidopsis and the Wc-1 (Crosthwaite et al., 1997) and VVD (Heintzen et al., 2001)circadian regulators of Neurospora, little is known about the function of these putativephotoreceptors. Notably, LOV domains are fused to a broad array of signal-output do-mains and range in size from the very small Neurospora VVD, containing little morethan a single LOV domain, to hybrid photoreceptor kinases in cyanobacteria con-taining upwards of 1800 amino acids (Crosson et al., 2003).

LOV proteins can be assigned to six functional categories based on their Pfam/Smart/COG annotation in the NCBI Conserved Domain Database (Marchler-Baueret al., 2003): 1) phototropins; 2) two-component signaling proteins; 3) STAS/sulfatetransport or sigma factor regulators; 4) GGDEF/EAL-phosphodiesterase/cyclases;5) G protein regulators (RGS proteins); and 6) proteins regulating circadian rhythm(Table 15.1 and Figure 15.6). With few exceptions, members of these categories havevery similar domain construction, with a LOV domain(s) in the amino-terminal re-gion of the protein followed by some type of signaling output (e.g. kinase, STAS,GGDEF) or DNA binding domain (e.g. GATA-type zinc finger) (Figure 15.6). Withthe exception of the hypothetical RGS protein of Magnaporthe grisea, LOV proteins ineukaryotes fall into either the phototropin or circadian regulator categories.

Bacterial LOV domains are fused to common prokaryotic signal output domainssuch as histidine kinases, response regulators, and GGDEF/EAL domains. Little isknown about the function of these bacterial proteins and how light may affect theiractivity. Indeed, the antisigma factor antagonist, YtvA, of Bacillus subtilis is the onlybacterial LOV protein with a known function (Akbar et al., 2001). However, no light-dependent activity in vivo or in vitro is known for YtvA. Understanding the biologicalrole of bacterial LOV proteins is one of the frontiers of blue light photobiology. Sur-prisingly, many of these putative photoreceptors are present in organisms that areboth non-photosynthetic and have no known behavioral response to light. It will beinteresting to see what role LOV proteins play in the biology of such species (e.g.Caulobacter crescentus, Bacillus subtilis, Listeria monocytogenes, and Magnetospirillummagnetotacticum). Importantly, the possibility that some prokaryotic LOV proteinsare not directly responding to light, but are sensing some other intra- or extracellularsignal cannot be ruled out.

How PAS domains transmit a signal to inter- and intramolecular partner domainsis a long-standing question in the study of PAS proteins. Recently, a picture of a con-served signaling pathway across the PAS β-sheet has begun to emerge (Erbel et al.,2003) that is supported by solution NMR work on oat phototropin1 LOV2 showinghelix undocking from the β-sheet in response to adduct formation (Harper et al.,2003). Future experiments that compare the biochemistry, structure, and dynamicsof full-length prokaryotic and eukaryotic LOV photoreceptors will allow us to identi-fy how LOV domains signal to their structurally- and functionally-variable partner do-mains and determine if the mode of signaling has been conserved over time.

15 LOV-Domain Structure, Dynamics, and Diversity

33315.5 LOV-Domain Diversity

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Acknowledgements

This work has benefited from countless discussions with Keith Moffat, Spencer An-derson, Jason Key, and Sudar Rajagopal regarding protein structure, function, anddynamics. My work on LOV domains would not have been possible without early helpfrom Winslow Briggs and John Christie. I also thank Kevin Gardner, John Kennis,and Trevor Swartz for many insightful discussions during the course of this work.Comments from Kevin Gardner and Shannon Harper greatly improved this manu-script.

15 LOV-Domain Structure, Dynamics, and Diversity

Table 15.1 LOV proteins present in GenBank as of January 2004. Hypothetical proteins are followed by their accession numbers.

1) Phototropin/phy3

Arabidopsis thaliana phot1 and phot2Oryza sativa phot1 and phot2Avena sativa phot1Zea mays phot1Adiantum capillus-veneris photPisum sativum phot1Chlamydomonas photSpinacia oleracea photVicia faba photAdiantum capillus-veneris phy3Onoclea sensibilis phy3Hypolepis punctata phy3Dryopteris f ilix-mas phy3

2) LOV Histidine Kinases and HybridHistidine Kinase/Response Regulators

Xanthomonas axonopodis AAM37406Xanthomonas campestris AAM41699Pseudomonas syringae AAO56389Caulobacter crescentus AAK22272Brucella melitensis AAL53921Brucella suis AAN33777M. magnetotacticum MAGN4020M. magnetotacticum MAGN5031N. aromaticivorans SARO2721Pirellula sp. 1 RB4511Thermosynechococcus elongatus LL1282Noctoc PCC7120 ALL2875Nostoc punctiforme NPUN0349Anabaena PCC7120 BAB74574

3) LOV Response Regulator

N. aromaticivorans SARO0132

4) LOV STAS Proteins

Bacillus subtilis YtvAOceanobacillus iheyensis BAC12544Listeria monocytogenes LMO0799Listeria innocua LIN0792

5) LOV GGDEF/EAL Proteins

Noctoc punctiforme NPUN5680Nostoc PCC7120 ALR3170Pseudomonas syringae PSYR0372Synechocystis P6803 SLR0359Ralstonia solanacearum RSP0254Anabaena PCC7120 BAB74869

6) Putative G Protein regulator

Magnaporthe grisea MG08735

7) LOV Circadian Regulators

Neurospora crassa VVDNeurospora crassa Wc-1Arabidopsis thaliana ZTLArabidopsis thaliana FKF1Arabidopsis thaliana LKP2

8) Other LOV Proteins

Arabidopsis thaliana AAC05351Magnaporthe grisea MG07517Pseudomonas putida PP4629Chlorof lexus aurantiacus CHLO3495Rhodobacter sphaeroides RSPH2966

335

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15 LOV-Domain Structure, Dynamics, and Diversity