scholarly journals Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

2021 ◽  
Vol 118 (20) ◽  
pp. e2024583118
Author(s):  
Takayuki Nagae ◽  
Masashi Unno ◽  
Taiki Koizumi ◽  
Yohei Miyanoiri ◽  
Tomotsumi Fujisawa ◽  
...  
Keyword(s):  
Structural Information ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Protonation State ◽  
Leaky Bucket ◽  
Control Of Gene Expression ◽  
Absorbing State ◽  
Chromatic Acclimation ◽  
Ring Nitrogen ◽  
Photosynthetic Antenna

Cyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. It is suggested that the absorption change of this subfamily is caused by the bilin C15-Z/C15-E photoisomerization and a subsequent change in the bilin protonation state. However, structural information and direct evidence of the bilin protonation state are lacking. Here, we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a “bucket” consisting of hydrophobic residues, in which the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A- through C-rings coplanar and the D-ring tilted. Three pyrrole nitrogens of the A- through C-rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen bonded in the β-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the bucket, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the “leaky bucket” structure functions as a proton exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.

scholarly journals Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

2020 ◽  
Author(s):  
Takayuki Nagae ◽  
Masashi Unno ◽  
Taiki Koizumi ◽  
Yohei Miyanoiri ◽  
Tomotsumi Fujisawa ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Mechanisms ◽  
Direct Evidence ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Nmr Analysis ◽  
Protonation State ◽  
Control Of Gene Expression ◽  
Absorbing State ◽  
Chromatic Acclimation

AbstractCyanobacteriochromes (CBCRs) are bilin-binding photosensors of the phytochrome superfamily that show remarkable spectral diversity. The green/red CBCR subfamily is important for regulating chromatic acclimation of photosynthetic antenna in cyanobacteria and is applied for optogenetic control of gene expression in synthetic biology. They are suggested to combine the bilin C15-Z/C15-E photoisomerization with a change in the bilin protonation state to drive their absorption changes. However, structural information and direct evidence of the bilin protonation state are lacking. Here we report a high-resolution (1.63Å) crystal structure of the bilin-binding domain of the chromatic acclimation sensor RcaE in the red-absorbing photoproduct state. The bilin is buried within a “pan” consisting of hydrophobic residues, where the bilin configuration/conformation is C5-Z,syn/C10-Z,syn/C15-E,syn with the A–C rings co-planar and the D-ring tilted. Three pyrrole nitrogens of the A–C rings are covered in the α-face with a hydrophobic lid of Leu249 influencing the bilin pKa, whereas they are directly hydrogen-bonded in the β-face with the carboxyl group of Glu217. Glu217 is further connected to a cluster of waters forming a hole in the pan, which are in exchange with solvent waters in molecular dynamics simulation. We propose that the “holey pan” structure functions as a proton-exit/influx pathway upon photoconversion. NMR analysis demonstrated that the four pyrrole nitrogen atoms are indeed fully protonated in the red-absorbing state, but one of them, most likely the B-ring nitrogen, is deprotonated in the green-absorbing state. These findings deepen our understanding of the diverse spectral tuning mechanisms present in CBCRs.Significance StatementGreen/red CBCRs are one of the most important CBCR subfamilies owing to their physiological roles in cyanobacteria phylum and optogenetic applications. They are known to utilize a change in the bilin protonation state to drive the marked change in green/red absorption, but the structural basis of the protochromic green/red photocycle are not well understood. Here, we have determined the crystal structure of the chromatic acclimation sensor RcaE of this subfamily in the photoproduct state, demonstrating a unique conformation of the bilin and its interacting residues. In addition, we provide direct evidence of the protonation state of the bilin via NMR analysis. These findings bring insight to our understanding of the molecular mechanisms underlying the spectral diversity of CBCRs.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Structural Basis for Linezolid Binding Site Rearrangement in the Staphylococcus aureus Ribosome

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Matthew J. Belousoff ◽  
Zohar Eyal ◽  
Mazdak Radjainia ◽  
Tofayel Ahmed ◽  
Rebecca S. Bamert ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Structural Information ◽  
Structural Basis ◽  
Common Mechanism ◽  
Resistance Mutations ◽  
Antimicrobial Drugs ◽  
Linezolid Resistance ◽  
Drug Modification ◽  
Antibiotic Resistance Mutations ◽  
Shed Light

ABSTRACT An unorthodox, surprising mechanism of resistance to the antibiotic linezolid was revealed by cryo-electron microscopy (cryo-EM) in the 70S ribosomes from a clinical isolate of Staphylococcus aureus. This high-resolution structural information demonstrated that a single amino acid deletion in ribosomal protein uL3 confers linezolid resistance despite being located 24 Å away from the linezolid binding pocket in the peptidyl-transferase center. The mutation induces a cascade of allosteric structural rearrangements of the rRNA that ultimately results in the alteration of the antibiotic binding site. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance. IMPORTANCE The growing burden on human health caused by various antibiotic resistance mutations now includes prevalent Staphylococcus aureus resistance to last-line antimicrobial drugs such as linezolid and daptomycin. Structure-informed drug modification represents a frontier with respect to designing advanced clinical therapies, but success in this strategy requires rapid, facile means to shed light on the structural basis for drug resistance (D. Brown, Nat Rev Drug Discov 14:821–832, 2015, https://doi.org/10.1038/nrd4675 ). Here, detailed structural information demonstrates that a common mechanism is at play in linezolid resistance and provides a step toward the redesign of oxazolidinone antibiotics, a strategy that could thwart known mechanisms of linezolid resistance.

Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

2017 ◽  
Vol 474 (20) ◽  
pp. 3373-3389 ◽  
Author(s):  
Dong-Dong Meng ◽  
Xi Liu ◽  
Sheng Dong ◽  
Ye-Fei Wang ◽  
Xiao-Qing Ma ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Glycoside Hydrolase ◽  
Complex Structure ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Glycoside Hydrolase Family ◽  
Substrate Selectivity ◽  
Glucose Residue ◽  
Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

scholarly journals Cryo-EM structure of the complete and ligand-saturated insulin receptor ectodomain

2019 ◽  
Author(s):  
Theresia Gutmann ◽  
Ingmar Schäfer ◽  
Chetan Poojari ◽  
Beate Brankatschk ◽  
Ilpo Vattulainen ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Binding Sites ◽  
Structural Model ◽  
Receptor Site ◽  
Insulin Binding ◽  
Proximal Region ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Structure Based Drug Design ◽  
Receptor Interactions

AbstractGlucose homeostasis and growth essentially depend on the peptide hormone insulin engaging its receptor. Despite biochemical and structural advances, a fundamental contradiction has persisted in the current understanding of insulin ligand–receptor interactions. While biochemistry predicts two distinct insulin binding sites, 1 and 2, recent structural analyses have only resolved site 1. Using a combined approach of cryo-EM and atomistic molecular dynamics simulation, we determined the structure of the entire dimeric insulin receptor ectodomain saturated with four insulin molecules. Complementing the previously described insulin–site 1 interaction, we present the first view of insulin bound to the discrete insulin receptor site 2. Insulin binding stabilizes the receptor ectodomain in a T-shaped conformation wherein the membrane-proximal domains converge and contact each other. These findings expand the current models of insulin binding to its receptor and of its regulation. In summary, we provide the structural basis enabling a comprehensive description of ligand–receptor interactions that ultimately will inform new approaches to structure-based drug design.In briefA cryo-EM structure of the complete insulin receptor ectodomain saturated with four insulin ligands is reported. The structural model of the insulin–insulin receptor complex adopts a T-shaped conformation, reveals two additional insulin-binding sites potentially involved in the initial interaction of insulin with its receptor, and resolves the membrane proximal region.

scholarly journals Flexibility of Short-Strand RNA in Aqueous Solution as Revealed by Molecular Dynamics Simulation: Are A-RNA and A´-RNA Distinct Conformational Structures?

2009 ◽  
Vol 62 (9) ◽  
pp. 1054 ◽  
Author(s):  
Defang Ouyang ◽  
Hong Zhang ◽  
Dirk-Peter Herten ◽  
Harendra S. Parekh ◽  
Sean C. Smith
Keyword(s):  
Molecular Dynamics ◽  
Aqueous Solution ◽  
Structural Information ◽  
Structural Parameters ◽  
Biological Molecules ◽  
Dynamics Simulation ◽  
Viral Gene ◽  
Conformational Structure ◽  
Crystal Forms ◽  
Gene Delivery Vectors

We use molecular dynamics simulations to compare the conformational structure and dynamics of a 21-base pair RNA sequence initially constructed according to the canonical A-RNA and A′-RNA forms in the presence of counterions and explicit water. Our study aims to add a dynamical perspective to the solid-state structural information that has been derived from X-ray data for these two characteristic forms of RNA. Analysis of the three main structural descriptors commonly used to differentiate between the two forms of RNA – namely major groove width, inclination and the number of base pairs in a helical twist – over a 30 ns simulation period reveals a flexible structure in aqueous solution with fluctuations in the values of these structural parameters encompassing the range between the two crystal forms and more. This provides evidence to suggest that the identification of distinct A-RNA and A′-RNA structures, while relevant in the crystalline form, may not be generally relevant in the context of RNA in the aqueous phase. The apparent structural flexibility observed in our simulations is likely to bear ramifications for the interactions of RNA with biological molecules (e.g. proteins) and non-biological molecules (e.g. non-viral gene delivery vectors).

A Stochastic Dynamics Simulation of Viscoelastic Properties of Polymer Blends: Intermolecular Interaction Effects

1998 ◽  
Vol 18 (1-2) ◽  
pp. 1-16
Author(s):  
Taehyung Kim ◽  
Kyoungsei Choi ◽  
Won Ho Jo
Keyword(s):  
Shear Rate ◽  
Polymer Blends ◽  
Intermolecular Interaction ◽  
Viscoelastic Properties ◽  
Stochastic Dynamics ◽  
Structural Information ◽  
Model Systems ◽  
Dynamics Simulation ◽  
Radius Of Gyration ◽  
Orientation Factor

Abstract Stochastic dynamics simulations were performed to investigate the viscoelastic properties of polymer blends. In this simulation, three model systems with different intermolecular interactions are used to examine the effect of intermolecular interaction on the viscoelastic properties of polymer blends. Structural information such as the radius of gyration, orientation factor and radial distribution function of polymers is calculated from computer simulations as a function of shear rate and then is related to simulated viscoelastic properties of polymer blends. The effect of intermolecular interaction on the viscosity becomes different depending upon the magnitude of shear rate. At lower shear rate regions, more attractive intermolecular interaction results in lower viscosity due to chain stretching. But, at higher shear rate regions, more attractive interaction results in higher viscosity due to more dense packing of chains induced by the intermolecular attraction.

scholarly journals Structural basis for transcription inhibition by E. coli SspA

2020 ◽  
Vol 48 (17) ◽  
pp. 9931-9942 ◽  
Author(s):  
Fulin Wang ◽  
Jing Shi ◽  
Dingwei He ◽  
Bei Tong ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Structural Information ◽  
Protein A ◽  
Acid Tolerance ◽  
In Vitro Transcription ◽  
Nucleotide Metabolism ◽  
Structural Basis ◽  
E Coli ◽  
Promoter Escape ◽  
Zinc Binding Domain

Abstract Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

scholarly journals Structural basis of homologous recombination

2019 ◽  
Vol 77 (1) ◽  
pp. 3-18 ◽  
Author(s):  
Yueru Sun ◽  
Thomas J. McCorvie ◽  
Luke A. Yates ◽  
Xiaodong Zhang
Keyword(s):  
Breast Cancer ◽  
Electron Microscopy ◽  
Homologous Recombination ◽  
Ataxia Telangiectasia ◽  
Structural Information ◽  
Homology Search ◽  
Structural Basis ◽  
Cancer Type ◽  
Dna Double Strand Breaks ◽  
Breast Cancer Type

AbstractHomologous recombination (HR) is a pathway to faithfully repair DNA double-strand breaks (DSBs). At the core of this pathway is a DNA recombinase, which, as a nucleoprotein filament on ssDNA, pairs with homologous DNA as a template to repair the damaged site. In eukaryotes Rad51 is the recombinase capable of carrying out essential steps including strand invasion, homology search on the sister chromatid and strand exchange. Importantly, a tightly regulated process involving many protein factors has evolved to ensure proper localisation of this DNA repair machinery and its correct timing within the cell cycle. Dysregulation of any of the proteins involved can result in unchecked DNA damage, leading to uncontrolled cell division and cancer. Indeed, many are tumour suppressors and are key targets in the development of new cancer therapies. Over the past 40 years, our structural and mechanistic understanding of homologous recombination has steadily increased with notable recent advancements due to the advances in single particle cryo electron microscopy. These have resulted in higher resolution structural models of the signalling proteins ATM (ataxia telangiectasia mutated), and ATR (ataxia telangiectasia and Rad3-related protein), along with various structures of Rad51. However, structural information of the other major players involved, such as BRCA1 (breast cancer type 1 susceptibility protein) and BRCA2 (breast cancer type 2 susceptibility protein), has been limited to crystal structures of isolated domains and low-resolution electron microscopy reconstructions of the full-length proteins. Here we summarise the current structural understanding of homologous recombination, focusing on key proteins in recruitment and signalling events as well as the mediators for the Rad51 recombinase.

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