Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice
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ABSTRACT:
SUBMITTER: Xiaoyu Guo
PROVIDER: S-SCDT-10_15252-EMBJ_2021110518 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:BACKGROUND:Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2 degrees C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life. RESULTS:We studied the reversible cold lability of E. coli Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo E. coli Trpase all apo forms of Trpase dissociated into dimers already at 25 degrees C and even further upon cooling to 2 degrees C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9A resolution. These apo mutants were found in an open conformation compared to the closed conformation found for P. vulgaris in its holo form. This conformational change is further supported by a high pressure study. CONCLUSION:We suggest that cold lability of E. coli Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of E. coli Trpase to dimers and P. vulgaris Trpase to monomers.
| S-EPMC2770544 | biostudies-literature
Project description:Somatostatin (SRIF) is a 14-residue peptide hormone synthesized in the hypothalamus and pancreatic islets. SRIF-14 and an N-terminally extended form, SRIF-28, are generated by the proteolytic processing of an approx. 102-residue precursor prosomatostatin (proSRIF) at a single set of paired basic residues (Arg-Lys) and at a monobasic (Arg) site respectively. Previous work in our laboratory demonstrated that the propeptide of SRIF mediates intracellular sorting; we suggested that this information resides in the prohormone structure. To identify putative sorting domains we have investigated structural features of recombinant anglerfish proSRIF-II purified from Escherichia coli. Two species of proSRIF-II were obtained: a monomeric form and a disulphide-linked dimer. CD analyses revealed that monomeric proSRIF-II lacks appreciable periodic secondary structure; however, on slow heating (2 degrees C/min) and cooling, it assumed a predominantly alpha-helical conformation. When subjected to a second heating-and-cooling cycle, the alpha-helical conformation was maintained. In contrast, the dimeric form of proSRIF-II was predominantly alpha-helical and its helicity did not increase in response to heating and recooling. Our results suggest that proSRIF-II might exist in several different folding intermediate states.
| S-EPMC1219689 | biostudies-other
Project description:Despite the large number of studies available on nicotinic acetylcholine receptors, a complete account of the mechanistic aspects of their gating transition in response to ligand binding still remains elusive. As a first step toward dissecting the transition mechanism by accelerated sampling techniques, we study the ligand-induced conformational changes of the acetylcholine binding protein (AChBP), a widely accepted model for the full receptor extracellular domain. Using unbiased Molecular Dynamics (MD) and Temperature Accelerated Molecular Dynamics (TAMD) simulations we investigate the AChBP transition between the apo and the agonist-bound state. In long standard MD simulations, both conformations of the native protein are stable, while the agonist-bound structure evolves toward the apo one if the orientation of few key sidechains in the orthosteric cavity is modified. Conversely, TAMD simulations initiated from the native conformations are able to produce the spontaneous transition. With respect to the modified conformations, TAMD accelerates the transition by at least a factor 10. The analysis of some specific residue-residue interactions points out that the transition mechanism is based on the disruption/formation of few key hydrogen bonds. Finally, while early events of ligand dissociation are observed already in standard MD, TAMD accelerates the ligand detachment and, at the highest TAMD effective temperature, it is able to produce a complete dissociation path in one AChBP subunit.
| S-EPMC3923797 | biostudies-literature
Project description:Temperature is a key factor in the growth and development of all organisms1,2. Plants have to interpret temperature fluctuations, over hourly to monthly timescales, to align their growth and development with the seasons. Much is known about how plants respond to acute thermal stresses3,4, but the mechanisms that integrate long-term temperature exposure remain unknown. The slow, winter-long upregulation of VERNALIZATION INSENSITIVE 3 (VIN3)5-7, a PHD protein that functions with Polycomb repressive complex 2 to epigenetically silence FLOWERING LOCUS C (FLC) during vernalization, is central to plants interpreting winter progression5,6,8-11. Here, by a forward genetic screen, we identify two dominant mutations of the transcription factor NTL8 that constitutively activate VIN3 expression and alter the slow VIN3 cold induction profile. In the wild type, the NTL8 protein accumulates slowly in the cold, and directly upregulates VIN3 transcription. Through combining computational simulation and experimental validation, we show that a major contributor to this slow accumulation is reduced NTL8 dilution due to slow growth at low temperatures. Temperature-dependent growth is thus exploited through protein dilution to provide the long-term thermosensory information for VIN3 upregulation. Indirect mechanisms involving temperature-dependent growth, in addition to direct thermosensing, may be widely relevant in long-term biological sensing of naturally fluctuating temperatures.
| S-EPMC7116785 | biostudies-literature
Project description:Cd(2+) binding at the bacterial photosynthetic reaction center (bRC) from Rhodobacter sphaeroides is known to inhibit proton transfer (PT) from bulk solvent to the secondary quinone Q(B). To elucidate this mechanism, we calculated the pK(a) for residues along the water channels connecting Q(B) with the stromal side based on the crystal structures of WT-bRC and Cd(2+)-bound bRC. Upon Cd(2+) binding, we observed the release of two protons from His-H126/128 at the Cd(2+) binding site and significant pK(a) shifts for residues along the PT pathways. Remarkably, Asp-L213 near Q(B), which is proposed to play a significant role in PT, resulted in a decrease in pK(a) upon Cd(2+) binding. The direct electrostatic influence of the Cd(2+)-positive charge on these pK(a) shifts was small. Instead, conformational changes of amino acid side chains induced electrostatically by Cd(2+) binding were the main mechanism for these pK(a) shifts. The long-range electrostatic influence over approximately 12 A between Cd(2+) and Asp-L213 is likely to originate from a set of Cd(2+)-induced successive reorientations of side chains (Asp-H124, His-H126, His-H128, Asp-H170, Glu-H173, Asp-M17, and Asp-L210), which propagate along the PT pathways as a "domino" effect.
| S-EPMC1283420 | biostudies-literature
Project description:BACKGROUND:Members of the periplasmic binding protein (PBP) superfamily are involved in transport and signaling processes in both prokaryotes and eukaryotes. Biological responses are typically mediated by ligand-induced conformational changes in which the binding event is coupled to a hinge-bending motion that brings together two domains in a closed form. In all PBP-mediated biological processes, downstream partners recognize the closed form of the protein. This motion has also been exploited in protein engineering experiments to construct biosensors that transduce ligand binding to a variety of physical signals. Understanding the mechanistic details of PBP conformational changes, both global (hinge bending, twisting, shear movements) and local (rotamer changes, backbone motion), therefore is not only important for understanding their biological function but also for protein engineering experiments. RESULTS:Here we present biochemical characterization and crystal structure determination of the periplasmic ribose-binding protein (RBP) from the hyperthermophile Thermotoga maritima in its ribose-bound and unliganded state. The T. maritima RBP (tmRBP) has 39% sequence identity and is considerably more resistant to thermal denaturation (app Tm value is 108 degrees C) than the mesophilic Escherichia coli homolog (ecRBP) (app Tm value is 56 degrees C). Polar ligand interactions and ligand-induced global conformational changes are conserved among ecRBP and tmRBP; however local structural rearrangements involving side-chain motions in the ligand-binding site are not conserved. CONCLUSION:Although the large-scale ligand-induced changes are mediated through similar regions, and are produced by similar backbone movements in tmRBP and ecRBP, the small-scale ligand-induced structural rearrangements differentiate the mesophile and thermophile. This suggests there are mechanistic differences in the manner by which these two proteins bind their ligands and are an example of how two structurally similar proteins utilize different mechanisms to form a ligand-bound state.
| S-EPMC2630998 | biostudies-literature
Project description:The non-visual arrestins, arrestin-2 and arrestin-3, belong to a small family of multifunctional cytosolic proteins. Non-visual arrestins interact with hundreds of G protein-coupled receptors (GPCRs) and regulate GPCR desensitization by binding active phosphorylated GPCRs and uncoupling them from heterotrimeric G proteins. Recently, non-visual arrestins have been shown to mediate G protein-independent signaling by serving as adaptors and scaffolds that assemble multiprotein complexes. By recruiting various partners, including trafficking and signaling proteins, directly to GPCRs, non-visual arrestins connect activated receptors to diverse signaling pathways. To investigate arrestin-mediated signaling, a structural understanding of arrestin activation and interaction with GPCRs is essential. Here we identified global and local conformational changes in the non-visual arrestins upon binding to the model GPCR rhodopsin. To detect conformational changes, pairs of spin labels were introduced into arrestin-2 and arrestin-3, and the interspin distances in the absence and presence of the receptor were measured by double electron electron resonance spectroscopy. Our data indicate that both non-visual arrestins undergo several conformational changes similar to arrestin-1, including the finger loop moving toward the predicted location of the receptor in the complex as well as the C-tail release upon receptor binding. The arrestin-2 results also suggest that there is no clam shell-like closure of the N- and C-domains and that the loop containing residue 136 (homolog of 139 in arrestin-1) has high flexibility in both free and receptor-bound states.
| S-EPMC4110305 | biostudies-literature
Project description:Ca(II) ions are crucial during proteolytic conversion of Factor XIII zymogen into the active enzyme Factor XIIIa. Factor XIII proteolyzed by thrombin or trypsin in the presence of 5 mM-EDTA resulted in rapid inactivation of transglutaminase activity. Factor XIIIa formed by thrombin or trypsin in the presence of 40 microM-Tb(III) ions, however, was indistinguishable from Factor XIIIa formed in the presence of 2-5 mM-Ca(II) ions with respect to molecular mass and transglutaminase activity. Thrombin treatment of Factor XIII in the presence of 1-5 microM-Tb(III) ions resulted in three fragments (76 kDa, 51 kDa and 19 kDa) with simultaneous loss of transglutaminase activity. Tb(III) ions at concentrations greater than 40 microM made platelet Factor XIII resistant to proteolysis by either thrombin or trypsin. Other lanthanide(III) ions [Ln(III) ions] tested [Ce(III), La(III) and Gd(III) ions] functioned similarly to Tb(III) ions during proteolytic activation of Factor XIII. Ln(III) ions (10-100 microM) were unable to replace the Ca(II) ions required for transglutaminase activity of Factor XIIIa. Tb(III) ions also inhibited in a non-competitive manner the transglutaminase activity of Factor XIIIa (Ki 71 microM) even when measured in the presence of 200-fold molar excess of Ca(II) ions. Factor XIII selectively bound to a Tb(III)-chelate affinity column, and could not be eluted by 100 mM-CaCl2. Binding of Tb(III) ions to Factor XIII was demonstrated by fluorescence emission due to Forster energy transfer. A 10(4)-fold molar excess of CaCl2, but not NaCl, partially quenched Tb(III) fluorescence. Low concentrations (5-20 microM) of Tb(III) ions also inhibited the binding of Factor XIII to des-A-fibrinogen by about 43%, whereas higher concentrations (40-100 microM) promoted binding. Conformational changes in Factor XIII consequent to the binding of Tb(III) ions could be responsible for the observed effects on protein structure and function.
| S-EPMC1135584 | biostudies-other
Project description:A computational docking strategy using multiple conformations of the target protein is discussed and evaluated. A series of low molecular weight, competitive, nonpeptide protein tyrosine phosphatase inhibitors are considered for which the x-ray crystallographic structures in complex with protein tyrosine phosphatase 1B (PTP1B) are known. To obtain a quantitative measure of the impact of conformational changes induced by the inhibitors, these were docked to the active site region of various structures of PTP1B using the docking program FlexX. Firstly, the inhibitors were docked to a PTP1B crystal structure cocrystallized with a hexapeptide. The estimated binding energies for various docking modes as well as the RMS differences between the docked compounds and the crystallographic structure were calculated. In this scenario the estimated binding energies were not predictive inasmuch as docking modes with low estimated binding energies corresponded to relatively large RMS differences when aligned with the corresponding crystal structure. Secondly, the inhibitors were docked to their parent protein structures in which they were cocrystallized. In this case, there was a good correlation between low predicted binding energy and a correct docking mode. Thirdly, to improve the predictability of the docking procedure in the general case, where only a single target protein structure is known, we evaluate an approach which takes possible protein side-chain conformational changes into account. Here, side chains exposed to the active site were considered in their allowed rotamer conformations and protein models containing all possible combinations of side-chain rotamers were generated. To evaluate which of these modeled active sites is the most likely binding site conformation for a certain inhibitor, the inhibitors were docked against all active site models. The receptor rotamer model corresponding to the lowest estimated binding energy is taken as the top candidate. Using this protocol, correct inhibitor binding modes could successfully be discriminated from proposed incorrect binding modes. Moreover, the ranking of the estimated ligand binding energies was in good agreement with experimentally observed binding affinities.
| S-EPMC1302794 | biostudies-literature
Project description:Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface stimulates association of CRT with LDL receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. The structural basis for this phenomenon is unknown. We studied the binding thermodynamics of the TSP1-CRT complex and the conformational changes in CRT induced by binding to TSP1 with combined binding free energy analysis, molecular dynamics simulation, and anisotropic network model restrained molecular dynamics simulation. Results showed that mutations of Lys 24 and Lys 32 in TSP1 to Ala and of amino acids 24-26 and 32-34 in CRT to Ala significantly weakened the binding of TSP1 and CRT, which is consistent with experimental results. Upon validation of the calculated binding affinity changes of the TSP1-CRT complex by mutations in key residues in TSP1 and CRT with the experimental results, we performed conformational analyses to understand the role of TSP1 binding to CRT in the induction of conformational changes in CRT. Conformational analyses showed that TSP1 binding to CRT resulted in a more "open" conformation and a significant rotational change for the CRT N-domain with respect to the CRT P-domain, which could expose the potential binding site(s) in CRT for binding to LRP1 to signal focal adhesion disassembly. Results offer structural insight into the role of TSP1 binding to CRT in CRT-induced focal adhesion disassembly.
| S-EPMC2943676 | biostudies-literature