scholarly journals Two clusters of surface-exposed amino acid residues enable high-affinity binding of retinal degeneration-3 (RD3) protein to retinal guanylyl cyclase

2020 ◽  
Vol 295 (31) ◽  
pp. 10781-10793 ◽  
Author(s):  
Igor V. Peshenko ◽  
Alexander M. Dizhoor
Keyword(s):  
Retinal Degeneration ◽  
Guanylyl Cyclase ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Hek293 Cells ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Retinal Guanylyl Cyclase ◽  
Functional Interface

Retinal degeneration-3 (RD3) protein protects photoreceptors from degeneration by preventing retinal guanylyl cyclase (RetGC) activation via calcium-sensing guanylyl cyclase–activating proteins (GCAP), and RD3 truncation causes severe congenital blindness in humans and other animals. The three-dimensional structure of RD3 has recently been established, but the molecular mechanisms of its inhibitory binding to RetGC remain unclear. Here, we report the results of probing 133 surface-exposed residues in RD3 by single substitutions and deletions to identify side chains that are critical for the inhibitory binding of RD3 to RetGC. We tested the effects of these substitutions and deletions in vitro by reconstituting purified RD3 variants with GCAP1-activated human RetGC1. Although the vast majority of the surface-exposed residues tolerated substitutions without loss of RD3's inhibitory activity, substitutions in two distinct narrow clusters located on the opposite sides of the molecule effectively suppressed RD3 binding to the cyclase. The first surface-exposed cluster included residues adjacent to Leu63 in the loop connecting helices 1 and 2. The second cluster surrounded Arg101 on a surface of helix 3. Single substitutions in those two clusters drastically, i.e. up to 245-fold, reduced the IC50 for the cyclase inhibition. Inactivation of the two binding sites completely disabled binding of RD3 to RetGC1 in living HEK293 cells. In contrast, deletion of 49 C-terminal residues did not affect the apparent affinity of RD3 for RetGC. Our findings identify the functional interface on RD3 required for its inhibitory binding to RetGC, a process essential for protecting photoreceptors from degeneration.

scholarly journals Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

2008 ◽  
Vol 190 (6) ◽  
pp. 2056-2064 ◽  
Author(s):  
Jonathan E. Ulmer ◽  
Yap Boum ◽  
Christopher D. Thouvenel ◽  
Hannu Myllykallio ◽  
Carol Hopkins Sibley
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Thymidylate Synthase ◽  
Three Dimensional ◽  
Pyrimidine Ring ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Catalytic Residue ◽  
Amino Acid Residues ◽  
Insight Into

ABSTRACT A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX7-8S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a ΔthyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X24 H69X25R95HRX7 S105XRYX90R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.

scholarly journals GUCY2D mutations in retinal guanylyl cyclase 1 provide biochemical reasons for dominant cone–rod dystrophy but not for stationary night blindness

2020 ◽  
Vol 295 (52) ◽  
pp. 18301-18315
Author(s):  
Igor V. Peshenko ◽  
Elena V. Olshevskaya ◽  
Alexander M. Dizhoor
Keyword(s):  
Guanylyl Cyclase ◽  
Night Blindness ◽  
Hek293 Cells ◽  
Dimerization Domain ◽  
Rods And Cones ◽  
Gene Coding ◽  
Rod Function ◽  
Retinal Guanylyl Cyclase ◽  
Leber’S Congenital Amaurosis

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

scholarly journals In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

2002 ◽  
Vol 184 (14) ◽  
pp. 4018-4024 ◽  
Author(s):  
Ulf Olsson ◽  
Annika Billberg ◽  
Sara Sjövall ◽  
Salam Al-Karadaghi ◽  
Mats Hansson
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure ◽  
Activity Measurements

ABSTRACT Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V max. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

scholarly journals A Microfluidic Probe Integrated Device for Spatiotemporal 3D Chemical Stimulation in Cells

Micromachines ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 691
Author(s):  
Kenta Shinha ◽  
Wataru Nihei ◽  
Hiroshi Kimura
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Chemical Stimulation ◽  
Dimensional Structure ◽  
Biological Research ◽  
Flow Profile ◽  
Novel Device ◽  
Biological Studies ◽  
Integrated Device

Numerous in vitro studies have been conducted in conventional static cell culture systems. However, most of the results represent an average response from a population of cells regardless of their local microenvironment. A microfluidic probe is a non-contact technology that has been widely used to perform local chemical stimulation within a restricted space, providing elaborated modulation and analysis of cellular responses within the microenvironment. Although microfluidic probes developed earlier have various potential applications, the two-dimensional structure can compromise their functionality and flexibility for practical use. In this study, we developed a three-dimensional microfluidic probe integrated device equipped with vertically oriented microchannels to overcome crucial challenges and tested the potential utility of the device in biological research. We demonstrated that the device tightly regulated spatial diffusion of a fluorescent molecule, and the flow profile predicted by simulation replicated the experimental results. Additionally, the device modulated the physiological Ca2+ response of cells within the restricted area by altering the local and temporal concentrations of biomolecules such as ATP. The novel device developed in this study may provide various applications for biological studies and contribute to further understanding of molecular mechanisms underlying cellular physiology.

scholarly journals In Silico Prediction, Molecular Docking and Dynamics Studies of Steroidal Alkaloids of Holarrhena pubescens Wall. ex G. Don to Guanylyl Cyclase C: Implications in Designing of Novel Antidiarrheal Therapeutic Strategies

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4147
Author(s):  
Neha Gupta ◽  
Saurav Kumar Choudhary ◽  
Neeta Bhagat ◽  
Muthusamy Karthikeyan ◽  
Archana Chaturvedi
Keyword(s):  
Molecular Docking ◽  
Amino Acid ◽  
Guanylyl Cyclase ◽  
Extracellular Domain ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Guanylyl Cyclase C ◽  
Amino Acid Residues ◽  
Lead Compounds

The binding of heat stable enterotoxin (STa) secreted by enterotoxigenic Escherichia coli (ETEC) to the extracellular domain of guanylyl cyclase c (ECDGC-C) causes activation of a signaling cascade, which ultimately results in watery diarrhea. We carried out this study with the objective of finding ligands that would interfere with the binding of STa on ECDGC-C. With this view in mind, we tested the biological activity of a alkaloid rich fraction of Holarrhena pubescens against ETEC under in vitro conditions. Since this fraction showed significant antibacterial activity against ETEC, we decided to test the screen binding affinity of nine compounds of steroidal alkaloid type from Holarrhena pubescens against extracellular domain (ECD) by molecular docking and identified three compounds with significant binding energy. Molecular dynamics simulations were performed for all the three lead compounds to establish the stability of their interaction with the target protein. Pharmacokinetics and toxicity profiling of these leads demonstrated that they possessed good drug-like properties. Furthermore, the ability of these leads to inhibit the binding of STa to ECD was evaluated. This was first done by identifying amino acid residues of ECDGC-C binding to STa by protein–protein docking. The results were matched with our molecular docking results. We report here that holadysenterine, one of the lead compounds that showed a strong affinity for the amino acid residues on ECDGC-C, also binds to STa. This suggests that holadysenterine has the potential to inhibit binding of STa on ECD and can be considered for future study, involving its validation through in vitro assays and animal model studies.

scholarly journals Nanofiber-Mâché Hollow Ball Mimicking the Three-Dimensional Structure of a Cyst

Polymers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 2273
Author(s):  
Wan-Ying Huang ◽  
Norichika Hashimoto ◽  
Ryuhei Kitai ◽  
Shin-ichiro Suye ◽  
Satoshi Fujita
Keyword(s):  
Three Dimensional ◽  
Hollow Structure ◽  
Dimensional Structure ◽  
The Novel ◽  
Fibrous Scaffold ◽  
Hydrogel Beads ◽  
Inner Surface ◽  
Intracranial Epidermoid ◽  
Hollow Ball

The occasional malignant transformation of intracranial epidermoid cysts into squamous cell carcinomas remains poorly understood; the development of an in vitro cyst model is urgently needed. For this purpose, we designed a hollow nanofiber sphere, the “nanofiber-mâché ball.” This hollow structure was fabricated by electrospinning nanofiber onto alginate hydrogel beads followed by dissolving the beads. A ball with approximately 230 mm3 inner volume provided a fibrous geometry mimicking the topography of the extracellular matrix. Two ducts located on opposite sides provided a route to exchange nutrients and waste. This resulted in a concentration gradient that induced oriented migration, in which seeded cells adhered randomly to the inner surface, formed a highly oriented structure, and then secreted a dense web of collagen fibrils. Circumferentially aligned fibers on the internal interface between the duct and hollow ball inhibited cells from migrating out of the interior, similar to a fish bottle trap. This structure helped to form an adepithelial layer on the inner surface. The novel nanofiber-mâché technique, using a millimeter-sized hollow fibrous scaffold, is excellently suited to investigating cyst physiology.

Characterization of an A-type cyclin-dependent kinase gene from Dendrobium candidum

Biologia ◽  
2012 ◽  
Vol 67 (2) ◽  
Author(s):  
Gang Zhang ◽  
Chao Song ◽  
Ming-Ming Zhao ◽  
Biao Li ◽  
Shun-Xing Guo
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Postembryonic Development ◽  
Kinase Domain ◽  
Dimensional Structure ◽  
Pcr Analysis ◽  
Kinase Gene ◽  
Multiple Sequence ◽  
Dendrobium Candidum ◽  
Rapid Amplification

AbstractCyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and postembryonic development of organisms. To better understand the molecular mechanisms of CDKs involved in embryogenesis regulation in the endangered medicinal plant Dendrobium candidum Wall. ex Lindl., a 1229-bp full-length cDNA of an A-type CDK gene, Denca;CDKA;1, was identified using 3′ rapid amplification of cDNA end (RACE) PCR. Denca;CDKA;1 was predicted to encode a 294 amino acid residue-long protein of 33.76 kDa with an isoelectric point of 7.72. The deduced Denca;CDKA;1 protein contained a conserved serine/threonine-protein kinase domain (S-TKc) and a canonical cyclinbinding “PSTAIRE” motif. Multiple sequence alignment indicated that members of CDKA family from various plants exhibited a high degree of sequence identity ranging from 82% to 93%. A neighbor-joining phylogenetic tree showed that Denca;CDKA;1 was clustered into the plant group and was distant from the animal and fungal groups. The modeled three-dimensional structure of Denca;CDKA;1 exhibited the similar functional structure of a fold consisting of β-sheets and α-helices joined by discontinuous random coils forming two relatively independent lobes. Quantitative real-time PCR analysis revealed that Denca;CDKA;1 transcripts were the most abundant in protocorm-like bodies with 4.76 fold, followed by that in roots (4.19 fold), seeds (2.57 fold), and stems (1.57 fold). This study characterized the novel Denca;CDKA;1 gene from D. candidum for the first time and the results will be useful for further functional determination of the gene.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Conformation and membrane interaction studies of the potent antimicrobial and anticancer peptide palustrin-Ca

2021 ◽  
Author(s):  
Patrick Brendan Timmons ◽  
Chandralal M Hewage
Keyword(s):  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Dynamics Simulation ◽  
Peptide Sequence ◽  
Helical Conformation ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Anticancer Activities ◽  
American Bullfrog ◽  
Α Helix

Palustrin-Ca (GFLDIIKDTGKEFAVKILNNLKCKLAGGCPP) is a host defense peptide with potent antimicrobial and anticancer activities, first isolated from the skin of the American bullfrog Lithobates catesbeianus. The peptide is 31 amino acid residues long, cationic and amphipathic. Two-dimensional NMR spectroscopy was employed to characterise its three-dimensional structure in a 50/50% water/2,2,2-trifluoroethanol-d3 mixture. The structure is defined by an α-helix that spans between Ile6-Ala26, and a cyclic disulphide bridged domain at the C-terminal end of the peptide sequence, between residues 23 and 29. A molecular dynamics simulation was employed to model the peptide's interactions with sodium dodecyl sulphate micelles, a widely used bacterial membrane-mimicking environment. Throughout the simulation, the peptide was found to maintain its α-helical conformation between residues Ile6-Ala26, while adopting a position parallel to the surface to micelle, which is energetically-favourable due to many hydrophobic and electrostatic contacts with the micelle.

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