LOV Domains in the Design of Photoresponsive Enzymes

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LOV Domains in the Design of Photoresponsive Enzymes Swantje Seifert and Susanne Brakmann*

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Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 4a, 44227 Dortmund, Germany ABSTRACT: In nature, a multitude of mechanisms have emerged for regulating biological processes and, specifically, protein activity. Light as a natural regulatory element is of outstanding interest for studying and modulating protein activity because it can be precisely applied with regard to a site of action, instant of time, or intensity. Naturally occurring photoresponsive proteins, predominantly those containing a light-oxygen-voltage (LOV) domain, have been characterized structurally and mechanistically and also conjugated to various proteins of interest. Immediate advantages of these new photoresponsive proteins such as genetic encoding, no requirement of chemical modification, and reversibility are paid for by difficulties in predicting the envisaged activity or type and site of domain fusion. In this article, we summarize recent advances and give a survey on currently available design concepts for engineering photoswitchable proteins.

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Engineering new regulatory mechanisms into a protein of interest requires that potential allosteric sites and amino acids involved in allosteric signal transduction are identified. Despite a tremendous collection of structural data (e.g., > 135 000 protein structures available in the Protein Data Bank; November 2017) and theoretical work that led to computational prediction methods,9 this is still challenging. During the past few years, much research has been dedicated to the development of empirical approaches by engineering proteins which may respond to chemical or optical input signals. In particular, optogenetic tools attracted much notice not only for studying and controlling the structure and function of isolated proteins12 but also for in vivo perturbing and deciphering complex cellular processes. Target systems range from binding of small molecules (e.g., inhibitors, metabolites) to reactions and reaction cascades (e.g., transcription−translation, RNA/ gene editing)13 and highly dynamic information processing networks that are necessary for organismal patterning (e.g., tissue or organ formation).14−16 Many approaches for engineering optical control of enzyme function involve photoresponsive proteins which are found in plants, algae, and bacteria for photosynthesis or photomorphogenesis. These proteins always utilize a cofactora small molecule that acts as a chromophore for harvesting light. Nature’s solution to the problem of engineering allosterically adjustable protein function led to modular systems having a photosensitive or sensor domain (= input module which usually interacts with a chromophore) and a f unctional or ef fector domain (= output module).

ellular metabolism is formed by an intricate network of highly interconnected biochemical reactions.1 On the basis of the human genome sequence and its annotations as well as published biochemical knowledge from five decades, our understanding of this network of more than 3500 reactions has been shaped through manual reconstructions2 and computational modeling.3 However, the questions of how and when an individual reaction takes place and how the multitude of reactions are coordinated in this network that maintains survival are still intriguingand far from being answered. Understanding intracellular regulation of enzymatic activity, e.g., by allosteric mechanisms, activation of zymogens, or reversible covalent modification, not only is central for our reception of “life” but also is of the utmost importance for the development of therapeutic approaches for malfunctioning networks, for the application of enzymes in man-made technologies, or for attempts to engineer a living system from the bottom up. Modulation or perturbation of protein function by allosteric regulation was discovered more than 50 years ago4,5 as a mechanism by which a signal from an external stimulus (e.g., noncovalent or covalent binding of a molecule to an allosteric binding site, or incidence of light at a sensor site) is transduced to a spatially distinct ef fector site of a protein (“remote control”).6,7 How chemical information is transmitted across a protein has been intensely studied and debated. In one of the present views, it is assumed thatbeyond discrete structural states and local interactions in a proteinstatistically correlated networks of amino acids play a major role.8 Addressing this subject from a more general perspective, it was discovered that proteins exist in ensembles of conformational states which might be associated with changes in protein dynamics and form entropic contributions to allostery. Hence, an allosteric signal might be propagated in a wave-like manner along evolutionarily conserved pathways.9−11 © XXXX American Chemical Society

Received: February 15, 2018 Accepted: June 15, 2018 Published: June 15, 2018 A

DOI: 10.1021/acschembio.8b00159 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Reviews

ACS Chemical Biology

Figure 1. (a) Structure of Avena sativa phototropin-1 (AsLOV2, dark state; PDB: 2V1A) showing the typical LOV domain fold with cofactor FMN bound in the central pocket. Picture generated using Pymol.24 (b) Schematic depiction of the LOV domain’s photocycle showing the involvement of a central cysteine residue.

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HOW DID NATURE DESIGN “PHOTOCONTROL OF ENZYME FUNCTION?” One of the natural design principles for light control of enzymatic activity is based on the linkage of light-induced changes of a photosensory domain with structural (steric, dynamic, electronic) changes in a tethered protein. Several classes of photosensory domains have evolved in nature which differ with respect to photochemistry and chromophor and, collectively, cover almost all wavelengths of visible light (e.g., phytochromes (chromophor: tetrapyrrole; 660 and 730 nm),17 cryptochromes (chromophor: pterin or flavin adenine dinucleotide (FAD); 380 or 450 nm),18 and phototropins (chromophor: flavin mononucleotide (FMN); 320−400 nm and 400−500 nm)).19 Much structural, biophysical, and biochemical research has focused on light-oxygen-voltage (LOV) photoreceptors, which form a photoresponsive subclass of the larger Per-ARNT-Sim (PAS) domain familiy of photosensors. They share a common fold with a central fivestranded antiparallel β-sheet (Aβ, Bβ, Gβ, Hβ, Iβ) and αhelical connectors (Cα, Dα, Eα, Fα), which form a pocket within which a flavin cofactor (FMN, FAD, or riboflavin; Figure 1) usually is bound.20,21 Typical LOV core domains encompass about 110 amino acids with a flavin-binding consensus GXNCRFLQ motif on helix Eα.22,23 The cysteine residue located here is directly involved in the LOV domain’s photocycle and central for signaling. Natural LOV proteins show a domain architecture usually having the N-terminally located sensor domain at variable distance to an effector domain; as yet, no natural LOV protein has been identified having its sensor domain inserted into the effector domain. A tremendous variety of effector domains has been found that ranges from kinases (histidine kinases, serine/ threonine kinases), nucleotide cyclases, sulfate transporter antiσ antagonists, and phosphodiesterases to DNA-binding proteins (zinc finger and helix−turn−helix (HTH) proteins).25 In the simplest case, LOV domains may be stand-alone (e.g., function as dimerizing domains), while in other cases they may be doubled or combined with further sensor domains, e.g., additional PAS domains (Figure 2). How does signaling proceed in such a diverse group of proteins? During the past two decades, the structural data of ∼50 LOV domains were recorded, some of which in light and dark states (Protein Data Bank, www.rcsb.org26). On the basis of these insights, the effects of photon absorption by LOV photosensors can be dissected into a sequence of processes

Figure 2. Exemplary domain architectures of photosensory proteins. PAS, Per-ARNT-Sim domain; HTH, helix−turn−helix motif; STAS, sulfate transporter antisigma factor antagonist.

(see also Möglich et al.):27 The incoming signal, absorbed light, initiates a chemical reaction during which a metastable covalent adduct is formed between a flavin cofactor (carbon atom 4a) and a conserved cysteine residue of the LOV domain. The reaction proceeds via excitation of FMN with blue light (maximal energy of 266 kJ mol−1 for photons of 450 nm wavelength)28 to a singlet state, FMN*, that rapidly interconverts to a triplet state, 3FMN, which then reacts with the sulfur of a neighbored cysteine side chain, resulting in the “light state” of the protein.20,23 This protein state having a covalent cofactor adduct at its core differs from the “dark state” with respect to steric and electronic properties (strain, degrees of freedom, intramolecular interactions) and, consequently, induces a series of eventually small structural alterations that influence both local and global environment. Rearrangements of protein structure can furthermore be accompanied by altered protein dynamics. Most studies focused on a set of prototypic LOV proteins, among these plant phototropin (e.g., Avena sativa AsLOV2),29,30 fungal photoreceptor Vivid (e.g., Neurospora crassa, VVD),31 photoresponsive bacterial DNAbinding protein (Erythrobacter litoralis EL222),32 Bacillus species σB regulator YtvA,33,34 and clock-associated proteins (e.g., Arabidopsis thaliana FKF-1).35 The effects observed with LOV domains involve rearrangements of secondary structural motifs located N- or C-terminally (Figure 3a) with respect to the cofactor binding site and encompass (1) order−disorder transitions (unfolding of helix Jα, plant phototropin),36 (2) rotational twisting (Jα helices in a LOV dimer, YtvA),37 (3) displacement of secondary structural elements (N-terminal cap, Ncap, in VVD),38 or (4) disruption of interaction surfaces with the Jα helix in EL222.39 These rearrangements translate to allosteric mechanisms within the linked effector domains B

DOI: 10.1021/acschembio.8b00159 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

that can be summarized as follows: (1) removal of steric strain and inhibition of effector activity, (2) reorientation of domains for enabling or increasing effector activity, and (3) homo- or heterodimerization for enabling protein−protein interactions, eventually of split effector proteins (Table 1 and Figure 3b).

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HOW TO DESIGN “SWITCHABLE ENZYMES” Crystal structure analyses together with chemical and photophysical studies have provided us with tremendous insight into the fundamental mechanisms of natural photosensory proteins. It is obvious to apply and expand nature’s design principles to any protein of interest in order to get access to lightdependent, remote control of its activity, particularly because a light signal can be applied with high spatial and temporal precision. What Has Been Achieved So Far? The idea of caging or uncaging of biomolecules that dates back to the 1970s (term coined by Hoffman et al.;44 reviewed, e.g., by Mayer and Heckel)45 raised the interest of many biochemistsprobably due to the notion that this type of switching directly targets enzymatic activity via steric blocking of its active site or binding surface. The light-induced unfolding of Jα helices found in plant phototropins has particularly been utilized for realizing the “caging concept” for artificial photoreceptors. Specifically the LOV2 domain of Avena sativa (AsLOV2) has been widely employed with a number of effector proteins, none of which have been associated with a LOV domain in a natural context. Among these are Rac1-GTPase,46 Rho GTPase,47 caspase,48 dihydrofolate reductase,8 Trp repressor protein,49 calcium channel regulators,50 transcription factors,51 split inteins,52 degrons,53 chromatin modifiers,54 Src kinase and GEFs,47 or diverse regulating peptides.55−59 Using an alternative concept, artificial photoreceptors have been designed in which the accessibility of an active interaction site is controlled by the formation or dissociation of intermolecular interactions. Examples encompass the lightinduced dissociation of domains using LOV2 fusion, e.g., with Cas9 (in vivo),60 receptor tyrosine kinase,61 or nitrilase;62 the homo- or heterodimerization of LOV-coupled transcription

Figure 3. (a) Structural alignment of LOV domains from different species. Gold, Avena sativa phototropin-1 Jα helix (AsLOV2, dark state; PDB: 2V1A); dark blue, Neurospora crassa VVD N-cap (light state; PDB: 2PDR); dark gray, Erythrobacter litoralis EL222 LOVHTH; medium blue, EL222 Jα helix (light state; PDB: 3P7N); light blue, Bacillus species σB regulator (YtvA, dark state; PDB: 2PR5). (b) Schematic overview of proposed modes of action of selected LOV domains (for a more comprehensive listing, see OptoBase, www. optobase.org).

Table 1. Prototypic Photosensory Domains and Their Proposed Modes of Action As Derived from X-ray Crystal Structure Analyses or Functional Analysis (FKF1) photosensory protein [UniProt ID] NPH1-1

photosensory domain [organism]

cofactor

structural data [PDB ID]

proposed mode of action

refs

phototropin-1 LOV2 (AsLOV2) [Avena sativa]

FMN

2V1A, 2V1B, 2V0U, 2V0W

order−disorder transition (Jα helix unfolding and undocking) releases effector into active conformation

29, 30, 36, 40

Vivid PAS protein/ Photoactive flavo-yellow protein [Neurospora crassa]

FMN, FAD

2PDR, 2PDT, 2PD7, 2PD8, 3RH8

structural rearrangement (Ncap displacement) leads to homodimerization

31, 38, 41

FMN

3P7N

disruption of interaction interface leads to homodimerization and DNA binding

32, 39

[Q2NB981]

Light-activated DNAbinding protein [Erythrobacter litoralis]

YtvA [O34627]

Blue-light photoreceptor [Bacillus subtilis]

FMN, FAD, riboflavin

2PR5, 2PR6

rotational twisting of (coiled coil) Jα helices turns effector 33, 34, into active conformation 42

ADO3 (FKF1)

Flavin-binding Kelch F-box protein 1 [Arabidopsis thaliana]

FMN

n.d.

heterodimerization with an interaction partner (A. thaliana: Gigantea)

[O490031] VVD [Q9C3Y61] EL222

[Q9C9W9]

C

35, 43

DOI: 10.1021/acschembio.8b00159 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Table 2. Examples of Photoresponsive Proteins That Have Been Designed Based on Rational or Computational Approaches design approach rational

SCA MD simulation

photoresponsive protein

effector domain

sensor domain

insertion/fusion type

refs

PA-Rac1 YF1 dCas9-RsLOV Opto-T7-RNAP LOV2-DHFR LOV-TRAP PiL[D24]

Rac1 FixL Cas9 T7-RNAP DHFR TrpR PKM2

AsLOV2 YtvA RsLOV VVD AsLOV2 AsLOV2 AsLOV2

N-terminal domain substitution position identified by domain scanning specific split position allosteric site allosteric site allosteric site

46, 77 37 60 66, 67 8 49, 70 75

Figure 4. Schematic representation of principles for the design of photoswitchable enzymes.

factors (EL222),63 promotor structures (VVD),64 and protein−protein interactions (FKF-1/Gigantea);65 and reassociation of a split T7 RNA polymerase66,67 as well as a set of newly developed photoswitches (iLID,68 LOVTRAP,69 TULIP70). More subtle concepts of allosteric regulation in photoresponsive proteins have rarely been implementedprobably because their effects are difficult to predict. In an exemplary study, light-dependent regulation was achieved by inserting the AsLOV “input module” into different surface positions of E. coli dehydrofolate reductase (DHFR; “output module”) previously predicted to be allosteric sites. Although moderate changes of DHFR activity were observed upon illumination, the strategy showed that modular allosteric networks can be connected to form new allosteric control. Thus, impressive progress has been achieved toward lightdependent intervention into selected enzymatic activities. However, general strategies for the engineering of this regulatory mechanism in multiple protein families are still under development. For every new protein of interest, the important initial question is how to find the ideal site for combining sensor and effector domain. Terminal fusion of sensory and effector domains directly follows nature’s design concept and has been shown to be a viable strategy for generating photoresponsiveness in many protein families. However, insertion of a LOV domain into an effector proteinas yet not recognized in natureis a complex undertaking because it requires knowledge of

boundary conditions such as catalytically relevant conformational transitions or allosterically communicating networks of amino acids that neither are readily available nor comprehensible. Some experience has been gained from substituting functional domains,37,71,72 or from inserting at split positionssites at which a protein can be cut into fragments which reassociate, possibly during joint binding of a substrate, to become functional full-length protein again.66,67 New sites suitable for domain insertion can either be identified by using screening approaches based on domain insertion scanning73,74 or by computational analysis of sequence and structural patterns. Rational design of photoactivatable proteins particularly benefits from applying computational methods: their potential in identifying conserved communication between amino acid residues (the “statistical signature of functional constraints”) by statistical coupling analysis (SCA),8,49,70 in using molecular dynamics simulations,75 analysis of coevolving residues,76 or combinations of methods such as dynamic coupling analysis and static contact mapping for the prediction of potential insertion sites based on surfaceexposed regions that are mechanically coupled to the active center of a protein47 (Table 2 and Figure 4).

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PERSPECTIVES The multitude of photoresponsive proteins that have been successfully designed during the past 10 years demonstrate a formidable versatility and utility of light-dependent control of enzymatic activity. Many challenges such as the prediction of D

DOI: 10.1021/acschembio.8b00159 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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Molecular Dynamics (MD) Simulation: a computational simulation technique for analyzing the structural motions of atoms, ions, and molecules with regard to conformational dynamics. MD simulation is often used for the modeling of biomacromolecules.

suitable insertion sites for light-responsible domains, disclosure of allosteric communication networks, and protein−protein interaction surfaces as well as chromophore interaction tuning are currently being tackled. Others such as handling of photochemical side reactions which may hit, for example, amino acids or nucleobases by intermediate reactive oxygen species (ROS), or photocontrol in vivo showing few (or no) intracellular side effects, will have to be considered in more detail. For the future it is expected that research in this field will significantly enlarge our mechanistic understanding of allosteric interaction networks and signal transduction, and provide us with modules and construction guidance for new photoresponsive enzymes. These enzymes could be reversibly switched “on” and “off” for applications ranging from organic synthesis to synthetic biology. Ideally, this know-how will also pave the way for switching more complex effector proteins (multiprotein complexes, receptors), which themselves undergo large conformational transitions for biological function. Then, a wealth of applications in biology, biochemistry, and medicine will be available!

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Susanne Brakmann: 0000-0002-9906-3735 Notes

The authors declare no competing financial interest.

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KEYWORDS Photoswitchable proteins: consist of a photosensory domain that is linked to an (enzymatic) effector domain. In response to the incidence of light, protein activity can be switched between “on” and “off” states, which result from reversible structural changes. Light-oxygen-voltage (LOV) domain: a photosensory subclass of the Per-ARNT-Sim (PAS) family of protein domains that detects blue light using a flavin cofactor. In most cases, LOV domains are N-terminally fused to signaling or regulatory domains. Photocycle: a sequence of structural changes or (elementary) chemical reactions that molecules undergo in response to changes in light intensity or wavelength. Flavin chromophore: flavins are blue-light sensitive chromophores found in LOV proteins where they are required for photon absorption. After excitation with light, a flavin chromophore (flavin, flavin adenine mononucleotide (FMN), or riboflavin) can react with a nearby cysteine to form a covalent bond which, in turn, induces reversible structural alterations of the protein. Split protein assembly: method for interrogating proximitydependent interactions of proteins or protein domains that is based on the fragmentation of a protein into parts which can reassemble and regain function. Jα helix: a conserved, amphipathic C-terminal secondary structure of most LOV domains which undergoes unfolding/folding and dissociation from/association with the domain’s core during the LOV photocycle. Statistical Coupling Analysis (SCA): a computational method for analyzing multiple protein sequence alignments with regard to groups of coevolving amino acid residues. E

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Reviews

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DOI: 10.1021/acschembio.8b00159 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Reviews

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DOI: 10.1021/acschembio.8b00159 ACS Chem. Biol. XXXX, XXX, XXX−XXX