Benchmarking and Analysis of Protein Docking Performance in Rosetta v3.2

128

Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. To prevent high-light induced damage, cyanobacteria have developed photoprotective mechanisms. One involves a photoactive carotenoid protein that decreases the transfer of excess energy to the reaction centers. This protein, the orange carotenoid protein (OCP), is present in most cyanobacterial strains; it is activated by high light conditions and able to dissipate excess energy at the site of the light-harvesting antennae, the phycobilisomes. Restoration of normal antenna capacity involves the fluorescence recovery protein (FRP). The FRP acts to dissociate the OCP from the phycobilisomes by accelerating the conversion of the active red OCP to the inactive orange form. We have determined the 3D crystal structure of the FRP at 2.5 Å resolution. Remarkably, the FRP is found in two very different conformational and oligomeric states in the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that the dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity. nonphotochemical quenching | Synechocystis L ight is vital for the survival and growth of photosynthetic organisms. In natural environments, these organisms are exposed to varying light conditions in addition to the day/night cycle. Too much exposure to light causes the formation of reactive oxygen species that damage the sensitive photochemical reaction centers, and thus, a careful regulation of energy flow is critical. Under low light conditions, an efficient energy collection by the antennae complexes is achieved, whereas under high light conditions, the excess energy has to be diverted from photosynthesis (1).Plants and cyanobacteria have evolved different ways to deal with excess energy arriving at the reaction centers. Higher plants and green algae contain antenna complexes consisting of transmembrane proteins that sense the acidification of the thylakoid lumen and react by switching from efficient energy collection to heat dissipation (1, 2). Cyanobacteria, however, contain antenna complexes called phycobilisomes, which are membrane-anchored and consist of phycobilin proteins. Instead of sensing the effect of high light through pH changes, cyanobacterial phycobilisomes are quenched by a protein capable of directly sensing high light conditions, the orange carotenoid protein (OCP). The 35 kDa OCP consists of two distinct domains that encompass a ketocarotenoid (3′-hydroxyechinenone) in an all trans conformation (3-6). Irradiance with high light changes the OCP from an inactive orange (OCP o ) to an active red form (OCP r ) that is capable of binding to the phycobilisomes to prevent excess energy from flowing to the reaction centers. ...

Section: Resultsmentioning

confidence: 99%

Crystal structure of the FRP and identification of the active site for modulation of OCP-mediated photoprotection in cyanobacteria

Sutter

Wilson

Leverenz

et al. 2013

128

“…To computationally assess the flexibility of the crenactin filaments observed in our experiments, protein-protein dockings were carried out using the Rosetta docking protocol, RosettaDock (25,26) (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

“…Global docking experiments for each minimized complex were carried out using the Rosetta Docking Protocol, called RosettaDock (25,26). For each complex, 10,000 structures were generated and ranked according to their binding energy as calculated with the Rosetta InterfaceAnalyzer.…”

Section: Methodsmentioning

confidence: 99%

Archaeal actin from a hyperthermophile forms a single-stranded filament

Braun

Orlova

Valegård

et al. 2015

The prokaryotic origins of the actin cytoskeleton have been firmly established, but it has become clear that the bacterial actins form a wide variety of different filaments, different both from each other and from eukaryotic F-actin. We have used electron cryomicroscopy (cryo-EM) to examine the filaments formed by the protein crenactin (a crenarchaeal actin) from Pyrobaculum calidifontis, an organism that grows optimally at 90°C. Although this protein only has ∼20% sequence identity with eukaryotic actin, phylogenetic analyses have placed it much closer to eukaryotic actin than any of the bacterial homologs. It has been assumed that the crenactin filament is double-stranded, like F-actin, in part because it would be hard to imagine how a single-stranded filament would be stable at such high temperatures. We show that not only is the crenactin filament single-stranded, but that it is remarkably similar to each of the two strands in F-actin. A large insertion in the crenactin sequence would prevent the formation of an F-actin-like double-stranded filament. Further, analysis of two existing crystal structures reveals six different subunit-subunit interfaces that are filament-like, but each is different from the others in terms of significant rotations. This variability in the subunit-subunit interface, seen at atomic resolution in crystals, can explain the large variability in the crenactin filaments observed by cryo-EM and helps to explain the variability in twist that has been observed for eukaryotic actin filaments.helical polymers | variable twist | cytoskeletal filaments | crenactin A ctin is one of the most highly conserved as well as abundant eukaryotic proteins. From chickens to humans, an evolutionary separation of ∼350 million years, there are no amino acid changes in the skeletal muscle isoform of actin (1). There are at least six different mammalian isoforms that are quite similar to each other, and all seem to have diverged from a common ancestral actin gene (2). In contrast, we now know that bacteria have actin-like proteins that share a common fold (3-5) but have vanishingly little sequence similarity both among themselves and to eukaryotic actin (6).Two recent crystal structures of a crenarchaeal actin, crenactin (7, 8), raise interesting questions about the structure of the crenactin filament and its evolutionary relationship to F-actin. In both crystals (with two different space groups) crenactin forms a single-stranded filament with eight subunits per ∼420-Å righthanded turn, with a rise and rotation per subunit, therefore, of ∼53 Å and 45°, respectively. In contrast, in F-actin there is a rise and rotation of ∼55 Å and ∼27°along each of the two long-pitch right-handed strands. It was stated (9) that outside of the crystal the crenactin filaments are double-stranded, based upon the suggestion (8) that a single-stranded filament would unlikely be stable and that power spectra from crenactin filaments showed a strong layer line at ∼1/(210 Å), half of the repeat in the crystals.We have been able to ...

“…An all-atom model of the chimera was built starting from the available high-resolution structures of the AsLOV2 (dark state) and PAH1 domains [Protein Data Bank (PDB) ID codes 2V1A and 2RMR] (33,34). An optimal relative orientation for the two domains was determined using the RosettaDock software (35). The program generates several conformations of the encounter complex using random orientations and selects those with low energy values.…”

Section: Methodsmentioning

confidence: 99%

Regulation of neural gene transcription by optogenetic inhibition of the RE1-silencing transcription factor

Paonessa

Criscuolo

Sacchetti

et al. 2015

Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa lightoxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na + -channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na + currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.