Structural and Evolutionary Analyses of PR-4 SUGARWINs Points to a Different Pattern of Protein Function

SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.

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IDENTIFICATION OF SELETIVE CLAUDIN CATION CHANNEL BLOCKERS

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.057 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S25-S26

Author(s):

Jingjing Ma ◽

Emma Wu ◽

Ye Li ◽

William Seibel ◽

Le Shen ◽

...

Keyword(s):

Small Molecules ◽

Homology Model ◽

Docking Studies ◽

Channel Blockers ◽

Binding Modes ◽

Barrier Dysfunction ◽

Epithelial Barrier Function ◽

Dynamics Simulations ◽

In Silico Models

Abstract Compromised epithelial barrier function is known to be associated with inflammatory bowel disease (IBD) and may contribute to disease development. One mechanism of barrier dysfunction is increased expression of paracellular tight junction ion and water channels formed by claudins. Claudin-2 and -15 are two such channels. We hypothesize that blocking these channels could be a viable therapeutic approach to treat diarrhea. In an effort to develop blockers of these channels, we turn to our previously developed and validated in silico models of claudin-15 (Samanta et al. 2018). We reasoned that compounds that can bind with the interior of claudin pores can limit paracellular water and ion flux. Thus, we used docking algorithms to search for putative small molecules that bind in the claudin-15 pore. AutoDock Vina was initially used to assess rigid docking using small compound databases. The small molecules were analyzed based on binding affinity to the pore and visualized using VMD for their potential blockage of the channel. Clusters of binding modes were identified based on the prominent interacting residues of the protein with the small molecules. We initially screened 10,500 compounds from within the UIC Centre for Drug Discovery and a cross-section of 10,000 compounds from the NCI open compound repository. This initial screen allowed us to identify 2 first-in-class selective claudin-15 blockers with efficacy in MDCK monolayers induced to express claudin-15 and in wildtype duodenum. Next, we screened the entire NCI open compound repository for additional molecules structurally related to our best initially identified molecule and this has allowed us to identify 13 additional molecules that increase TER of claudin-15 expressing MDCK monolayers by 90–160%. Additionally, these molecules possess similar structural components that will be collected in a fragment library and explored through molecular dynamics simulations. We also developed a claudin-2 homology model on which we are performing docking studies and in vitro measurements, which we expect will result in similar candidate ligands for blocking claudin-2. Our study will provide important insight into the role of claudin-dependent cation permeability in fundamental physiology, which we believe will lead to the utility of claudin blockers as a novel and much needed approach to treat diseases such as IBD.

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Novel Dominant KCNQ2 Exon 7 Partial In-Frame Duplication in a Complex Epileptic and Neurodevelopmental Delay Syndrome

International Journal of Molecular Sciences ◽

10.3390/ijms21124447 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4447

Author(s):

Pedro A. Lazo ◽

Juan L. García ◽

Paulino Gómez-Puertas ◽

Íñigo Marcos-Alcalde ◽

Cesar Arjona ◽

...

Keyword(s):

Protein Function ◽

De Novo ◽

3D Structure ◽

Unknown Origin ◽

Unknown Etiology ◽

Neurodevelopmental Delay ◽

Calmodulin Binding ◽

Whole Exome ◽

Dynamics Simulations ◽

Frame Duplication

Complex neurodevelopmental syndromes frequently have an unknown etiology, in which genetic factors play a pathogenic role. This study utilizes whole-exome sequencing (WES) to examine four members of a family with a son presenting, since birth, with epileptic-like crises, combined with cerebral palsy, severe neuromotor and developmental delay, dystonic tetraparexia, axonal motor affectation, and hyper-excitability of unknown origin. The WES study detected within the patient a de novo heterozygous in-frame duplication of thirty-six nucleotides within exon 7 of the human KCNQ2 gene. This insertion duplicates the first twelve amino acids of the calmodulin binding site I. Molecular dynamics simulations of this KCNQ2 peptide duplication, modelled on the 3D structure of the KCNQ2 protein, suggest that the duplication may lead to the dysregulation of calcium inhibition of this protein function.

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Wheat TaLr35PR2 gene is required for Lr35-mediated adult plant resistance against leaf rust fungus

Functional Plant Biology ◽

10.1071/fp18340 ◽

2020 ◽

Vol 47 (1) ◽

pp. 26

Author(s):

Fang Liang ◽

Xiong Du ◽

Jiarui Zhang ◽

Xiaoying Li ◽

Fei Wang ◽

...

Keyword(s):

Leaf Rust ◽

Fungal Pathogens ◽

Developmental Stages ◽

Rust Fungus ◽

Expression Patterns ◽

Plant Defence ◽

Adult Plant ◽

Isogenic Line ◽

Critical Function ◽

Pathogenesis Related Protein

In this study we analysed the expression patterns of TaLr35PR2 and confirmed its role in Lr35-mediated adult resistance to leaf rust fungus. β-1,3-glucanase, a pathogenesis-related protein, has a critical function in plant defence response against fungal pathogens. We previously described the full-length gene TaLr35PR2, which encodes a protein exhibiting amino acid and structural similarity to β-1,3-glucanase, in the wheat near-isogenic line TcLr35 (GenBank accession number DQ294235.1). This work aimed to further assess TaLr35PR2 expression patterns and function in Lr35-mediated adult resistance to Puccinia triticina. Immunoblot was performed to demonstrate that TaLr35PR2 expression was triggered early by P. triticina, with expression levels markedly elevated in incompatible interaction compared with those in compatible one. Additionally, TaLr35PR2 accumulation steadily increased and overtly peaked after challenge with P. triticina through the various developmental stages of TcLr35 wheat, and remaining at similar levels after mock inoculation. Furthermore, TaLr35PR2 expression was significantly reduced in barley stripe mosaic virus (BSMV)-induced gene knockdown plants, in which pathological assessment revealed that TaLr35PR2-silenced plants was obviously susceptible to leaf rust fungus compared with wild-type TcLr35, indicating that Lr35-mediated resistance to leaf rust was diminished. These findings strongly suggest that TaLr35PR2 is involved in Lr35-mediated wheat defence against the leaf rust pathogen.

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Molecular dynamics simulations of the interaction of wild type and mutant human CYP2J2 with polyunsaturated fatty acids

BMC Research Notes ◽

10.1186/s13104-019-4797-8 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

K. K. Abelak ◽

D. Bishop-Bailey ◽

I. Nobeli

Keyword(s):

Molecular Dynamics ◽

Arachidonic Acid ◽

Molecular Dynamics Simulations ◽

Molecular Mechanisms ◽

Homology Model ◽

Cytochrome P450 Enzyme ◽

Wild Type ◽

Substrate Preferences ◽

P450 Enzyme ◽

Dynamics Simulations

Abstract Objectives The data presented here is part of a study that was aimed at characterizing the molecular mechanisms of polyunsaturated fatty acid metabolism by CYP2J2, the main cytochrome P450 enzyme active in the human cardiovasculature. This part comprises the molecular dynamics simulations of the binding of three eicosanoid substrates to wild type and mutant forms of the enzyme. These simulations were carried out with the aim of dissecting the importance of individual residues in the active site and the roles they might play in dictating the binding and catalytic specificity exhibited by CYP2J2. Data description The data comprise: (a) a new homology model of CYP2J2, (b) a number of predicted low-energy complexes of CYP2J2 with arachidonic acid, docosahexaenoic acid and eicosapentaenoic acid, produced with molecular docking and (c) a series of molecular dynamics simulations of the wild type and four mutants interacting with arachidonic acid as well as simulations of the wild type interacting with the two other eicosanoid ligands. The simulations may be helpful in identifying the determinants of substrate specificity of this enzyme and in unraveling the role of individual mutations on its function. They may also help guide the generation of mutants with altered substrate preferences.

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Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2

Nucleic Acids Research ◽

10.1093/nar/gkt1119 ◽

2013 ◽

Vol 42 (4) ◽

pp. 2725-2735 ◽

Cited By ~ 7

Author(s):

Ronnie P.-A. Berntsson ◽

Richard Odegrip ◽

Wilhelmina Sehlén ◽

Karin Skaar ◽

Linda M. Svensson ◽

...

Keyword(s):

Dna Binding ◽

Protein Function ◽

Specific Binding ◽

Gene Products ◽

Structural Determinants ◽

Bacteriophage P2 ◽

Defective Prophage ◽

Key Residues ◽

Functional Analyses ◽

Insight Into

Abstract The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

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Extracellular Cation Binding Pocket Is Essential For Ion Conduction Of OsHKT2;2

10.1101/471003 ◽

2018 ◽

Author(s):

Janin Riedelsberger ◽

Ariela Vergara-Jaque ◽

Miguel Piñeros ◽

Ingo Dreyer ◽

Wendy González

Keyword(s):

Protein Interactions ◽

Mutual Effect ◽

Homology Model ◽

Binding Pocket ◽

Cation Binding ◽

Ion Binding ◽

Salt Bridges ◽

Extracellular Medium ◽

Dynamics Simulations ◽

Sodium And Potassium

AbstractHKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity, and performed molecular dynamics simulations in the presence of sodium and potassium ions. By analyzing ion-protein interactions, we identified a cation binding pocket on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion binding pocket and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results.

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Prediction of fluorophore brightness in designed mini fluorescence activating proteins

10.1101/2021.07.21.452510 ◽

2021 ◽

Author(s):

Emma R. Hostetter ◽

Jeffrey R. Keyes ◽

Ivy Poon ◽

Justin P. Nguyen ◽

Jacob Nite ◽

...

Keyword(s):

Protein Function ◽

De Novo ◽

Energy Landscape ◽

Three Dimensional ◽

Computational Design ◽

Dimensional Structure ◽

Bond Rotation ◽

Beta Barrel ◽

Angle Rotation ◽

Dynamics Simulations

The de novo computational design of proteins with predefined three-dimensional structure is becoming much more routine due to advancements both in force fields and algorithms. However, creating designs with functions beyond folding is more challenging. In that regard, the recent design of small beta barrel proteins that activate the fluorescence of an exogenous small molecule chromophore (DFHBI) is noteworthy. These proteins, termed mini Fluorescence Activating Proteins (mFAPs), have been shown increase the brightness of the chromophore more than 100-fold upon binding to the designed ligand pocket. The design process created a large library of variants with different brightness levels but gave no rational explanation for why one variant was brighter than another. Here we use quantum mechanics and molecular dynamics simulations to investigate how molecular flexibility in the ground and excited states influences brightness. We show that the ability of the protein to resist dihedral angle rotation of the chromophore is critical for predicting brightness. Our simulations suggest that the mFAP/DFHBI complex has a rough energy landscape, requiring extensive ground-state sampling to achieve converged predictions of excited-state kinetics. While computationally demanding, this roughness suggests that mFAP protein function can be enhanced by reshaping the energy landscape towards states that better resist DFHBI bond rotation.

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Towards Protein Function Prediction Based on Molecular Dynamics Simulations

Biophysical Journal ◽

10.1016/j.bpj.2020.11.696 ◽

2021 ◽

Vol 120 (3) ◽

pp. 80a

Author(s):

Nicolai Kozlowski ◽

Malte Schäffner ◽

Andreas Volkhardt ◽

Helmut Grubmuller

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Protein Function ◽

Protein Function Prediction ◽

Function Prediction ◽

Dynamics Simulations

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SNP2SIM: A modular workflow for standardizing molecular simulation and functional analysis of protein variants

10.1101/457192 ◽

2018 ◽

Author(s):

Matthew D. McCoy ◽

Vikram Shivakumar ◽

Sridhar Nimmagadda ◽

Mohsin Saleet Jafri ◽

Subha Madhavan

Keyword(s):

Molecular Dynamics ◽

Protein Function ◽

Molecular Mechanisms ◽

Cancer Genomics ◽

Atomic Scale ◽

Protein Variant ◽

Docking Simulations ◽

Small Molecule Ligands ◽

Dynamics Simulations ◽

The Impact

AbstractMolecular simulations are used to provide insight into protein structure and function, and have the potential to provide important context when predicting the impact of sequence variation on protein function. In addition to understanding molecular mechanisms and interactions on the atomic scale, translational applications of those approaches include drug screening, development of novel molecular therapies, and treatment planning when selecting targeted therapies. Supporting the continued development of these applications, we have developed the SNP2SIM workflow generates reproducible molecular dynamics and molecular docking simulations for downstream functional variant analysis. Three modules execute molecular dynamics simulations of solvated protein variant structures, analyze the resulting trajectories for unique structural conformations, and bind small molecule ligands to representative variant scaffolds. In addition to availability as a command line workflow, SNP2SIM modules are also available as individual apps on the Seven Bridges Cancer Genomics Cloud.

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Causality, Transfer Entropy and Allosteric Communication Landscapes in Proteins with Harmonic Interactions

10.1101/084764 ◽

2016 ◽

Cited By ~ 1

Author(s):

Aysima Hacisuleyman ◽

Burak Erman

Keyword(s):

Network Model ◽

Protein Function ◽

Information Transfer ◽

Protein Activity ◽

Additional Information ◽

Gaussian Network Model ◽

Allosteric Communication ◽

Rapid Characterization ◽

Dynamics Simulations ◽

Gaussian Network

AbstractA fast and approximate method of generating allosteric communication landscapes is presented by using Schreiber's entropy transfer concept in combination with the Gaussian Network Model of proteins. Predictions of the model and the allosteric communication landscapes generated show that information transfer in proteins does not necessarily take place along a single path, but through an ensemble of pathways. The model emphasizes that knowledge of entropy only is not sufficient for determining allosteric communication and additional information based on time delayed correlations has to be introduced, which leads to the presence of causality in proteins. The model provides a simple tool for mapping entropy sink-source relations into pairs of residues. Residues that should be manipulated to control protein activity may be determined with this approach. This should be of great importance for allosteric drug design and for understanding the effects of mutations on protein function. The model is applied to determine allosteric communication in two proteins, Ubiquitin and Pyruvate Kinase. Predictions are in agreement with detailed molecular dynamics simulations and experimental evidence.SignificanceProteins perform their function by an exchange of information within themselves and with their environments through correlated fluctuations of their atoms. Fluctuations of one atom may drive the fluctuations of another. Information transmitted in this way leads to allosteric communication which is described as the process in which action at one site of the protein is transmitted to another site at which the protein performs its activity. Disruption of allosteric communication by mutation for example leads to disease. The present paper incorporates information theoretic concepts into the well known Gaussian Network Model of proteins and allows for rapid characterization of allosteric communication landscapes for normal functioning as well as malfunctioning proteins.

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