The primary photochemistry is similar among the flavin‐bound sensory domains of light–oxygen–voltage (LOV) photoreceptors, where upon blue‐light illumination a covalent adduct is formed on the ...microseconds time scale between the flavin chromophore and a strictly conserved cysteine residue. In contrast, the adduct‐state decay kinetics vary from seconds to days or longer. The molecular basis for this variation among structurally conserved LOV domains is not fully understood. Here, we selected PpSB2‐LOV, a fast‐cycling (τrec 3.5 min, 20 °C) short LOV protein from Pseudomonas putida that shares 67% sequence identity with a slow‐cycling (τrec 2467 min, 20 °C) homologous protein PpSB1‐LOV. Based on the crystal structure of the PpSB2‐LOV in the dark state reported here, we used a comparative approach, in which we combined structure and sequence information with molecular dynamic (MD) simulations to address the mechanistic basis for the vastly different adduct‐state lifetimes in the two homologous proteins. MD simulations pointed toward dynamically distinct structural region, which were subsequently targeted by site‐directed mutagenesis of PpSB2‐LOV, where we introduced single‐ and multisite substitutions exchanging them with the corresponding residues from PpSB1‐LOV. Collectively, the data presented identify key amino acids on the Aβ‐Bβ, Eα‐Fα loops, and the Fα helix, such as E27 and I66, that play a decisive role in determining the adduct lifetime. Our results additionally suggest a correlation between the solvent accessibility of the chromophore pocket and adduct‐state lifetime. The presented results add to our understanding of LOV signaling and will have important implications in tuning the signaling behavior (on/off kinetics) of LOV‐based optogenetic tools.
The photocycle of light–oxygen–voltage (LOV) domains involves blue‐light‐triggered adduct formation between the bound flavin chromophore and a cysteine residue. LOV proteins show considerable variation in the lifetime of the adduct state. Here, we used a comparative approach selecting two homologous LOV proteins originating from Pseudomonas putida: a fast‐cycling PpSB2‐LOV (~ 3.5 min) and a slow‐cycling PpSB1‐LOV (~ 42 h) to investigate the mechanistic basis for the very different lifetimes of the adduct states.
Light–Oxygen–Voltage (LOV) domains represent the photo-responsive domains of various blue-light photoreceptor proteins and are widely distributed in plants, algae, fungi, and bacteria. Here, we ...report the dark-state crystal structure of PpSB1-LOV, a slow-reverting short LOV protein from Pseudomonas putida that is remarkably different from our previously published “fully light-adapted” structure 1. A direct comparison of the two structures provides insight into the light-activated signaling mechanism. Major structural differences involve a~11Å movement of the C terminus in helix Jα, ~4Å movement of Hβ–Iβ loop, disruption of hydrogen bonds in the dimer interface, and a~29° rotation of chain-B relative to chain-A as compared to the light-state dimer. Both crystal structures and solution NMR data are suggestive of the key roles of a conserved glutamine Q116 and the N-cap region consisting of A′α–Aβ loop and the A′α helix in controlling the light-activated conformational changes. The activation mechanism proposed here for the PpSB1-LOV supports a rotary switch mechanism and provides insights into the signal propagation mechanism in naturally existing and artificial LOV-based, two-component systems and regulators.
Display omitted
•Comparison of crystal structures of PpSB1-LOV in dark and light states•Dimer interface and the C-terminal Jα-helix show major structural rearrangements.•A~29° rotation between the two protein chains gated by light•Extensive NMR solution studies reveal light-induced conformational changes.•We propose a rotary switch mechanism for the activation.
•Transposition reactions are ubiquitous and involve complex DNA rearrangements.•Transposase-DNA complex structures reveal key mechanistic principles.•DNA bending, unwinding and melting promote all ...steps of transposase function.•DNA distortions serve universal roles in transposition and related cellular processes.
Transposases move discrete pieces of DNA between genomic locations and had a profound impact on evolution. They drove the emergence of important biological functions and are the most frequent proteins encoded in modern genomes. Yet, the molecular principles of their actions have remained largely unclear. Here we review recent structural studies of transposase-DNA complexes and related cellular machineries, which provided unmatched mechanistic insights. We highlight how transposases introduce major DNA twists and kinks at various stages of their reaction and discuss the functional impact of these astounding DNA acrobatics on several aspects of transposition. By comparison with distantly related DNA recombination systems, we propose that forcing DNA into unnatural shapes may be a general strategy to drive rearrangements forward.
Light-Oxygen-Voltage (LOV) proteins show considerable variation in the lifetime of the adduct state. Here, we used a comparative approach selecting two homologous LOV proteins originating from ...Pseudomonas fluorescens – SBW25-LOV and Pf5-LOV. At 37°C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. The cartoon representation on the left shows the light-dependent global structural changes in the dimeric short LOV proteins of Pseudomonadaceae, such as the rotation of the core domains relative to each other and the increased distances at the N- and C-terminal junctions.
Display omitted
•Short LOV (Light, Oxygen, Voltage) proteins from Pseudomonas fluorescens.•Crystal structures of SBW25-LOV (fully light-adapted), and Pf5-LOV (dark) state.•Light-induced rotation of core domains and splaying of N- and C-terminal helices.•Three non-conserved residues identified as crucial for tuning dark recovery kinetics.•Signal transduction mechanisms among Pseudomonadaceae LOV proteins is conserved.
Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A′α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.
Unique features of Light-Oxygen-Voltage (LOV) proteins like relatively small size (~12-19 kDa), inherent modularity, highly-tunable photocycle and oxygen-independent fluorescence have lately been ...exploited for the generation of optical tools. Structures of LOV domains reported so far contain a flavin chromophore per protein molecule. Here we report two new findings on the short LOV protein W619_1-LOV from Pseudomonas putida. First, the apo-state crystal structure of W619_1-LOV at 2.5 Å resolution reveals conformational rearrangements in the secondary structure elements lining the chromophore pocket including elongation of the Fα helix, shortening of the Eα-Fα loop and partial unfolding of the Eα helix. Second, the apo W619_1-LOV protein binds both natural and structurally modified flavin chromophores. Remarkably different photophysical and photochemical properties of W619_1-LOV bound to 7-methyl-8-chloro-riboflavin (8-Cl-RF) and lumichrome imply application of these variants as novel optical tools as they offer advantages such as no adduct state formation, and a broader choice of wavelengths for in vitro studies.
PDF
