Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCP^O) into the red-shifted, active state (OCP^R) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCP^R is converted to OCP^O and dissociates from PBs; however, the mode and site of OCP^R/FRP interactions remain elusive. Recently, we have introduced the purple OCP^W288A mutant as a competent model for the signaling state OCP^R (Sluchanko et al., Biochim Biophys Acta 1858:1–11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCP^W288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCP^O, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCP^O, a substantial compaction of the OCP^W288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCP^W288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.     

Picosecond kinetics of light harvesting and photoprotective quenching in wild-type and mutant phycobilisomes isolated from the cyanobacterium Synechocystis PCC 6803

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.     

Structural insight into the mechanism of epothilone A bound to beta-tubulin and its mutants at Arg282Gln and Thr274Ile

Epothilone A (EpoA) is under investigation as an antitumor agent. To provide better understanding of the activity of EpoA against cancers, by theoretical studies such as using docking method, molecular dynamics simulation and density functional theory calculations, we identify several key residues located on β-tubulin as the active sites to establish an active pocket responsible for interaction with EpoA. Eight residues (Arg276, Asp224, Asp26, His227, Glu27, Glu22, Thr274, and Met363) are identified as the active sites to form the active pocket on β-tubulin. The interaction energy is predicted to be -121.3?kJ/mol between EpoA and β-tubulin. In the mutant of β-tubulin at Thr274Ile, three residues (Arg359, Glu27, and His227) are identified as the active sites for the binding of EpoA. In the mutant of β-tubulin at Arg282Gln, three residues (Arg276, Lys19, and His227) serve as the active sites. The interaction energy is reduced to -77.2?kJ/mol between EpoA and Arg282Gln mutant and to -50.2?kJ/mol between EpoA and Thr274Ile mutant. The strong interaction with β-tubulin is significant to EpoA's activity against cancer cells. When β-tubulin is mutated either at Arg282Gln or at Thr274Ile, the decreased strength of interaction explains the activity reduced for EpoA. Therefore, this work shows that the structural basis of the active pocket plays an important role in regulating the activity for EpoA with a Taxol-like mechanism of action to be promoted as an antitumor agent.     

Engineering the photoactive orange carotenoid protein with redox-controllable structural dynamics and photoprotective function

Photosynthesis requires various photoprotective mechanisms for survival of organisms in high light. In cyanobacteria exposed to high light, the Orange Carotenoid Protein (OCP) is reversibly photoswitched from the orange (OCP^O) to the red (OCP^R) form, the latter binds to the antenna (phycobilisomes, PBs) and quenches its overexcitation. OCP^R accumulation implicates restructuring of a compact dark-adapted OCP^O state including detachment of the N-terminal extension (NTE) and separation of protein domains, which is reversed by interaction with the Fluorescence Recovery Protein (FRP). OCP phototransformation supposedly occurs via an intermediate characterized by an OCP^R-like absorption spectrum and an OCP^O-like protein structure, but the hierarchy of steps remains debatable. Here, we devise and analyze an OCP variant with the NTE trapped on the C-terminal domain (CTD) via an engineered disulfide bridge (OCP_CC). NTE trapping preserves OCP photocycling within the compact protein structure but precludes functional interaction with PBs and especially FRP, which is completely restored upon reduction of the disulfide bridge. Non-interacting with the dark-adapted oxidized OCP_CC, FRP binds reduced OCP_CC nearly as efficiently as OCP^O devoid of the NTE, suggesting that the low-affinity FRP binding to OCP^O is realized via NTE displacement. The low efficiency of excitation energy transfer in complexes between PBs and oxidized OCP_CC indicates that OCP_CC binds to PBs in an orientation suboptimal for quenching PBs fluorescence. Our approach supports the presence of the OCP^R-like intermediate in the OCP photocycle and shows effective uncoupling of spectral changes from functional OCP photoactivation, enabling redox control of its structural dynamics and function.     

Features of protein−protein interactions in the cyanobacterial photoprotection mechanism

Photoprotective mechanisms of cyanobacteria are characterized by several features associated with the structure of their water-soluble antenna complexes–the phycobilisomes (PBs). During energy transfer from PBs to chlorophyll of photosystem reaction centers, the “energy funnel” principle is realized, which regulates energy flux due to the specialized interaction of the PBs core with a quenching molecule capable of effectively dissipating electron excitation energy into heat. The role of the quencher is performed by ketocarotenoid within the photoactive orange carotenoid protein (OCP), which is also a sensor for light flux. At a high level of insolation, OCP is reversibly photoactivated, and this is accompanied by a sig- nificant change in its structure and spectral characteristics. Such conformational changes open the possibility for pro- tein–protein interactions between OCP and the PBs core (i.e., activation of photoprotection mechanisms) or the fluores- cence recovery protein. Even though OCP was discovered in 1981, little was known about the conformation of its active form until recently, as well as about the properties of homologs of its N and C domains. Studies carried out during recent years have made a breakthrough in understanding of the structural-functional organization of OCP and have enabled discovery of new aspects of the regulation of photoprotection processes in cyanobacteria. This review focuses on aspects of protein–pro- tein interactions between the main participants of photoprotection reactions and on certain properties of representatives of newly discovered families of OCP homologs.     

The N-terminal juxtamembrane segment of the V1a vasopressin receptor provides two independent epitopes required for high-affinity agonist binding and signaling

It is fundamentally important to define how agonist-receptor interaction differs from antagonist-receptor interaction. The V1a vasopressin receptor (V1aR) is a member of the neurohypophysial hormone subfamily of G protein-coupled receptors. Using alanine-scanning mutagenesis of the N-terminal juxtamembrane segment of the V1aR, we now establish that Glu54 (1.35) is critical for arginine vasopressin binding. The mutant [E54A]V1aR exhibited decreased arginine vasopressin affinity (1700-fold) and disrupted signaling, but antagonist binding was unaffected. Mutation of Glu54 had an almost identical pharmacological effect as mutation of Arg46, raising the possibility that agonist binding required a mutual interaction between Glu54 and Arg46. The role of these two charged residues was investigated by 1) substituting Glu54; 2) inserting additional Glu/Arg in transmembrane helix (TM) 1; 3) repositioning the Glu/Arg in TM1; and 4) characterizing the reciprocal mutant [R46E/E54R]V1aR. We conclude that 1) the positive/negative charges need to be precisely positioned in this N terminus/TM1 segment; and 2) Glu54 and Arg46 function independently, providing two discrete epitopes required for high-affinity agonist binding and signaling. This study explains why Glu and Arg, part of an -R(X3)L/V(X3)E(X3)L- motif, are conserved at these loci throughout this G protein-coupled receptor subfamily and provides molecular insight into key differences between agonist and antagonist binding requirements.     

Essential role of two tyrosines and two tryptophans on the photoprotection activity of the Orange Carotenoid Protein

Photosynthetic organisms have developed photoprotective mechanisms to protect themselves from lethal high light intensities. One of these mechanisms involves the dissipation of excess absorbed light energy into heat. In cyanobacteria, light activation of a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), binding a keto carotenoid, is the key inducer of this mechanism. Blue-green light absorption triggers structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here we report the role in photoconversion and photoprotection of individual conserved tyrosines and tryptophans surrounding the rings of the carotenoid. Our results demonstrate that the interaction between the keto group of the carotenoid and Tyr201 and Trp288 is essential for OCP photoactivity. In addition, these amino acids are responsible for carotenoid affinity and specificity. We have already demonstrated that the aromatic character of Tyr44 and Trp110 interacting with the hydroxyl ring is critical. Here we show that the replacement of Tyr44 by Ser affects the stability of the red form avoiding its accumulation at any temperature, while Trp110Ser is affected in the energy necessary to the orange to red conversion and in the interaction with the antenna. Collectively our data support the idea that the red form is essential for photoprotection but not sufficient. Specific conformational changes occurring in the protein seem to be critical to the events leading to energy dissipation.     

The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry

As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures — from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 μmol photons m^− 2 s^− 1. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Colipase residues Glu64 and Arg65 are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles

Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting PTL at the oil-water interface. The x-ray structure of the colipase.PTL complex supports this model. In the x-ray structure, colipase has a hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu(45)/Asp(89) and Glu(64)/Arg(65). To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu(64)/Arg(65) binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL.colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase.PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu(64)/Arg(65) binding site.     

Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli

The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.     

Localization of von willebrand factor-binding sites for platelet glycoprotein Ib and botrocetin by charged-to-alanine scanning mutagenesis

At sites of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion through binding to platelet glycoprotein Ib (GPIb). Previous studies identified clusters of charged residues within VWF domain A1 that were involved in binding GPIb or botrocetin. The contribution of 28 specific residues within these clusters was analyzed by mutating single amino acids to alanine. Binding to a panel of six conformation-dependent monoclonal antibodies was decreased by mutations at Asp(514), Asp(520), Arg(552), and Arg(611) (numbered from the N-terminal Ser of the mature processed VWF), suggesting that these residues are necessary for domain A1 folding. Binding of (125)I-botrocetin was decreased by mutations at Arg(629), Arg(632), Arg(636), and Lys(667). Ristocetin-induced and botrocetin-induced binding to GPIb both were decreased by mutations at Lys(599), Arg(629), and Arg(632); among this group the K599A mutant was unique because (125)I-botrocetin binding was normal, suggesting that Lys(599) interacts directly with GPIb. Ristocetin and botrocetin actions on VWF were dissociated readily by mutagenesis. Ristocetin-induced binding to GPIb was reduced selectively by substitutions at positions Lys(534), Arg(571), Lys(572), Glu(596), Glu(613), Arg(616), Glu(626), and Lys(642), whereas botrocetin-induced binding to GPIb was decreased selectively by mutations at Arg(636) and Lys(667). The binding of monoclonal antibody B724 involved Lys(660) and Arg(663), and this antibody inhibits (125)I-botrocetin binding to VWF. The crystal structure of the A1 domain suggests that the botrocetin-binding site overlaps the monoclonal antibody B724 epitope on helix 5 and spans helices 4 and 5. The binding of botrocetin also activates the nearby VWF-binding site for GPIb that involves Lys(599) on helix 3.     

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10^?5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

     

26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis

To explore the structure essential for the catalysis in 26 kDa endochitinase from barley seeds, we calculated theoretical pKa values of the ionizable groups based on the crystal structure, and then the roles of ionizable side chains located near the catalytic residue were examined by site-directed mutagenesis. The pKa value calculated for Arg215, which is located at the bottom of the catalytic cleft, is abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. No enzymatic activity was found in the Arg215-mutated chitinase (R215A) produced by the Escherichia coli expression system. The transition temperature of thermal unfolding (T(m)) of R215A was lower than that of the wild type protein by about 6.2 degrees C. In the crystal structure, the Arg215 side chain is in close proximity to the Glu203 side chain, whose theoretical pKa value was found to be abnormally low (-2.4), suggesting that these side chains may interact with each other. Mutation of Glu203 to alanine (E203A) completely eliminated the enzymatic activity and impaired the thermal stability (deltaT(m) = 6.4 degrees C) of the enzyme. Substrate binding ability was also affected by the Glu203 mutation. These data clearly demonstrate that the Arg215 side chain interacts with the Glu203 side chain to stabilize the conformation of the catalytic cleft. A similar interaction network was previously found in chitosanase from Streptomyces sp. N174 [Fukamizo et al. (2000) J. Biol. Chem. 275, 25633-25640]; hence, this type of interaction seems to be at least partly conserved in the catalytic cleft of other glycosyl hydrolases.     

Electrostatic stabilization of a thermophilic cold shock protein.

The cold shock protein Bc-Csp from the thermophile Bacillus caldolyticus differs from its mesophilic homolog Bs-CspB from Bacillus subtilis by 15.8 kJ mol(-1) in the Gibbs free energy of denaturation (DeltaG(D)). The two proteins vary in sequence at 12 positions but only two of them, Arg3 and Leu66 of Bc-Csp, which replace Glu3 and Glu66 of Bs-CspB, are responsible for the additional stability of Bc-Csp. These two positions are near the ends of the protein chain, but close to each other in the three-dimensional structure. The Glu3Arg exchange alone changed the stability by more than 11 kJ mol(-1). Here, we elucidated the molecular origins of the stability difference between the two proteins by a mutational analysis. Electrostatic contributions to stability were characterized by measuring the thermodynamic stabilities of many variants as a function of salt concentration. Double and triple mutant analyses indicate that the stabilization by the Glu3Arg exchange originates from three sources. Improved hydrophobic interactions of the aliphatic moiety of Arg3 contribute about 4 kJ mol(-1). Another 4 kJ mol(-1) is gained from the relief of a pairwise electrostatic repulsion between Glu3 and Glu66, as in the mesophilic protein, and 3 kJ mol(-1) originate from a general electrostatic stabilization by the positive charge of Arg3, which is not caused by a pairwise interaction. Mutations of all potential partners for an ion pair within a radius of 10 A around Arg3 had only marginal effects on stability. The Glu3-->Arg3 charge reversal thus optimizes ionic interactions at the protein surface by both local and global effects. However, it cannot convert the coulombic repulsion with another Glu residue into a corresponding attraction. Avoidance of unfavorable coulombic repulsions is probably a much simpler route to thermostability than the creation of stabilizing surface ion pairs, which can form only at the expense of conformational entropy.     

Insilico analysis and molecular docking of resuscitation promoting factor B (RpfB) protein of Mycobacterium tuberculosis

Invulnerability of Mycobacterium tuberculosis to various drugs and its persistency has stood as a hurdle in the race against eradication of the pathogenecity of the bacteria. Identification of novel antituberculosis compounds is highly demanding as the available drugs are resistant. The ability of the bacteria to surpass the body''s defenses and adapt itself to survive for disease reactivation is contributed by secreted proteins called resuscitating promoting factors (Rpfs). These factors aid in virulence and resuscitation from dormancy of the bacteria. Sequence analysis of RpfB was performed and compounds were first screened for toxicity and high-throughput virtual screening eliminating the toxic compounds. To understand the mechanism of ligand binding and interaction, molecular docking was performed for the compounds passing through the filter resulting with better docking studies predicting the possible binding mode of the inhibitors to the protein. Of all the active residues the binding conformation shows that residues Arg194, Arg196, Glu242, and Asn244 of the RpfB protein play vital role in the enzyme activity and interacts with the ligands. Promising compounds have been identified in the current study, thus holding promise for design of antituberculosis drugs.     

The highly conserved negatively charged Glu141 and Asp145 of the G-protein-coupled receptor RXFP3 interact with the highly conserved positively charged arginine residues of relaxin-3

Relaxin-3 is a newly identified insulin/relaxin superfamily peptide that plays a putative role in the regulation of food intake and stress response by activating its cognate G-protein-coupled receptor RXFP3. Relaxin-3 has three highly conserved arginine residues, B12Arg, B16Arg and B26Arg. We speculated that these positively charged arginines may interact with certain negatively charged residues of RXFP3. To test this hypothesis, we first replaced the negatively charged residues in the extracellular domain of RXFP3 with arginine, respectively. Receptor activation assays showed that arginine replacement of Glu141 or Asp145, especially Glu141, significantly decreased the sensitivity of RXFP3 to wild-type relaxin-3. In contrast, arginine replacement of other negatively charged extracellular residues had little effect. Thus, we deduced that Glu141 and Asp145, locating at the extracellular end of the second transmembrane domain, played a critical role in the interaction of RXFP3 with relaxin-3. To identify the ligand residues interacting with the negatively charged EXXXD motif of RXFP3, we replaced the three conserved arginines of relaxin-3 with negatively charged glutamate or aspartate, respectively. The mutant relaxin-3s retained the native structure, but their binding and activation potencies towards wild-type RXFP3 were decreased significantly. The compensatory effects of the mutant relaxin-3s towards mutant RXFP3s suggested two probable interaction pairs during ligand–receptor interaction: Glu141 of RXFP3 interacted with B26Arg of relaxin-3, meanwhile Asp145 of RXFP3 interacted with both B12Arg and B16Arg of relaxin-3. Based on these results, we proposed a relaxin-3/RXFP3 interaction model that shed new light on the interaction mechanism of the relaxin family peptides with their receptors.     

Glutamate-64, a newly identified residue of the functionally conserved electron-sharing network contributes to catalysis and structural integrity of glutathione transferases

In Anopheles dirus glutathione transferase D3-3, position 64 is occupied by a functionally conserved glutamate residue, which interacts directly with the gamma-glutamate moiety of GSH (glutathione) as part of an electron-sharing network present in all soluble GSTs (glutathione transferases). Primary sequence alignment of all GST classes suggests that Glu64 is one of a few residues that is functionally conserved in the GST superfamily. Available crystal structures as well as consideration of the property of the equivalent residue at position 64, acidic or polar, suggest that the GST electron-sharing motif can be divided into two types. Electrostatic interaction between the GSH glutamyl and carboxylic Glu64, as well as with Arg66 and Asp100, was observed to extend the electron-sharing motif identified previously. Glu64 contributes to the catalytic function of this motif and the 'base-assisted deprotonation' that are essential for GSH ionization during catalysis. Moreover, this residue also appears to affect multiple steps in the enzyme catalytic strategy, including binding of GSH, nucleophilic attack by thiolate at the electrophilic centre and product formation, probably through active-site packing effects. Replacement with non-functionally-conserved amino acids alters initial packing or folding by favouring aggregation during heterologous expression. Thermodynamic and reactivation in vitro analysis indicated that Glu64 also contributes to the initial folding pathway and overall structural stability. Therefore Glu64 also appears to impact upon catalysis through roles in both initial folding and structural maintenance.     

ApcD, ApcF and ApcE are not required for the Orange Carotenoid Protein related phycobilisome fluorescence quenching in the cyanobacterium Synechocystis PCC 6803

In cyanobacteria, strong blue-green light induces a photoprotective mechanism involving an increase of energy thermal dissipation at the level of phycobilisome (PB), the cyanobacterial antenna. This leads to a decrease of the energy arriving to the reaction centers. The photoactive Orange Carotenoid Protein (OCP) has an essential role in this mechanism. The binding of the red photoactivated OCP to the core of the PB triggers energy and PB fluorescence quenching. The core of PBs is constituted of allophycocyanin trimers emitting at 660 or 680nm. ApcD, ApcF and ApcE are the responsible of the 680nm emission. In this work, the role of these terminal emitters in the photoprotective mechanism was studied. Single and double Synechocystis PCC 6803 mutants, in which the apcD or/and apcF genes were absent, were constructed. The Cys190 of ApcE which binds the phycocyanobilin was replaced by a Ser. The mutated ApcE attached an unusual chromophore emitting at 710nm. The activated OCP was able to induce the photoprotective mechanism in all the mutants. Moreover, in vitro reconstitution experiments showed similar amplitude and rates of fluorescence quenching. Our results demonstrated that ApcD, ApcF and ApcE are not required for the OCP-related fluorescence quenching and they strongly suggested that the site of quenching is one of the APC trimers emitting at 660nm. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Residue 19 of the parathyroid hormone: structural consequences

Residue 19 of the parathyroid hormone (PTH) has been shown to play an important role in both binding to and activation of the PTH receptor; specifically, Arg(19)-containing analogues have improved biological function over similar Glu(19) peptides [Shimizu et al. (2002) Biochemistry 41, 13224-13233]. Additionally the juxtamembrane portion of the receptor is involved in the different biological responses. Here, we determine the conformational preferences of PTH analogues to provide a structural basis for their biological actions. On the basis of circular dichroism results, the Arg(19) --> Glu(19) mutations within the context of both PTH(1-20) and PTH(1-34) analogues lead to increases in helix content, ranging from a 8-15% increase. High-resolution structures as determined by (1)H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the difference between Arg(19) and Glu(19)-PTH(1-20), particularly with the extent and stability of the C-terminal helix. The Arg(19)-containing analogue has a well defined, stable alpha-helix from Ser(4)-Arg(19), while the Glu(19) analogue is less ordered at the C-terminus. On the basis of these observations, we propose that position 19 of PTH(1-20) must be alpha-helical for optimal interaction with the juxtamembrane portion of the receptor. This mode of binding extends the current view of PTH binding (indeed ligand binding for all class B GPCRs), which invokes a bihelical ligand with the C-terminus of the ligand interacting with the N-terminus of the receptor (responsible for binding) and the N-terminus of the ligand interacting with the seven-helical bundle (leading to receptor activation).