Crystal Structure of NADPH-Dependent Methylglyoxal Reductase Gre2 from Candida Albicans

Gre2 is a key enzyme in the methylglyoxal detoxification pathway; it uses NADPH or NADH as an electron donor to reduce the cytotoxic methylglyoxal to lactaldehyde. This enzyme is a member of the short-chain dehydrogenase/reductase (SDR) superfamily whose members catalyze this type of reaction with a broad range of substrates. To elucidate the structural features, we determined the crystal structures of the NADPH-dependent methylglyoxal reductase Gre2 from Candida albicans (CaGre2) for both the apo-form and NADPH-complexed form at resolutions of 2.8 and 3.02 Å, respectively. The CaGre2 structure is composed of two distinct domains: the N-terminal cofactor-binding domain and the C-terminal substrate-binding domain. Extensive comparison of CaGre2 with its homologous structures reveals conformational changes in α12 and β3′ of the NADPH-complex forms. This study may provide insights into the structural and functional variation of SDR family proteins.

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An enzyme captured in two conformational states: crystal structure ofS-adenosyl-L-homocysteine hydrolase fromBradyrhizobium elkanii

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715018659 ◽

2015 ◽

Vol 71 (12) ◽

pp. 2422-2432 ◽

Cited By ~ 6

Author(s):

Tomasz Manszewski ◽

Kriti Singh ◽

Barbara Imiolczyk ◽

Mariusz Jaskolski

Keyword(s):

Crystal Structure ◽

Enzymatic Reaction ◽

Binding Domain ◽

Dimerization Domain ◽

Biologically Relevant ◽

Cofactor Binding ◽

Enzymatic Regulation ◽

Ligand Free ◽

Substrate Binding Domain ◽

Methyl Group Transfer

S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation ofS-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM,S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase fromBradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Å resolution, showing the domain organization (substrate-binding domain, NAD+cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.

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Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714005422 ◽

2014 ◽

Vol 70 (5) ◽

pp. 1484-1490 ◽

Cited By ~ 12

Author(s):

Sally Dempster ◽

Stephen Harper ◽

John E. Moses ◽

Ingrid Dreveny

Keyword(s):

Lactate Dehydrogenase ◽

Conformational Changes ◽

Active Site ◽

Anaerobic Respiration ◽

Crystal Form ◽

Binary Complex ◽

Cofactor Binding ◽

Key Enzyme ◽

Crystallization Conditions

Lactate dehydrogenase A (LDH-A) is a key enzyme in anaerobic respiration that is predominantly found in skeletal muscle and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH. LDH-A is overexpressed in many tumours and has therefore emerged as an attractive target for anticancer drug discovery. Crystal structures of human LDH-A in the presence of inhibitors have been described, but currently no structures of the apo or binary NADH-bound forms are available for any mammalian LDH-A. Here, the apo structure of human LDH-A was solved at a resolution of 2.1 Å in space groupP4122. The active-site loop adopts an open conformation and the packing and crystallization conditions suggest that the crystal form is suitable for soaking experiments. The soaking potential was assessed with the cofactor NADH, which yielded a ligand-bound crystal structure in the absence of any inhibitors. The structures show that NADH binding induces small conformational changes in the active-site loop and an adjacent helix. A comparison with other eukaryotic apo LDH structures reveals the conservation of intra-loop interactions. The structures provide novel insight into cofactor binding and provide the foundation for soaking experiments with fragments and inhibitors.

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Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1619843114 ◽

2017 ◽

Vol 114 (23) ◽

pp. 6040-6045 ◽

Cited By ~ 9

Author(s):

Soumit Sankar Mandal ◽

Dale R. Merz ◽

Maximilian Buchsteiner ◽

Ruxandra I. Dima ◽

Matthias Rief ◽

...

Keyword(s):

Single Molecule ◽

Conformational Changes ◽

Structural Changes ◽

Substrate Binding ◽

Protein Structures ◽

Binding Domain ◽

Flexible Region ◽

Structural Parts ◽

The Individual ◽

Substrate Binding Domain

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD’s ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.

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Glycosaminoglycans induce conformational change in the SARS-CoV-2 Spike S1 Receptor Binding Domain

10.1101/2020.04.29.068767 ◽

2020 ◽

Cited By ~ 4

Author(s):

Courtney J. Mycroft-West ◽

Dunhao Su ◽

Yong Li ◽

Scott E. Guimond ◽

Timothy R. Rudd ◽

...

Keyword(s):

Conformational Change ◽

Receptor Binding ◽

Conformational Changes ◽

Heparan Sulphate ◽

Antiviral Agents ◽

Structural Features ◽

Binding Domain ◽

Microbial Pathogens ◽

Future Research ◽

Receptor Binding Domain

AbstractThe glycosaminoglycan (GAG) class of polysaccharides are utilised by a plethora of microbial pathogens as receptors for adherence and invasion. The GAG heparin prevents infection by a range of viruses when added exogenously, including the S-associated coronavirus strain HSR1 and more recently we have demonstrated that heparin can block cellular invasion by SARS-CoV-2. Heparin has found widespread clinical use as anticoagulant drug and this molecule is routinely used as a proxy for the GAG, heparan sulphate (HS), a structural analogue located on the cell surface, which is a known receptor for viral invasion. Previous work has demonstrated that unfractionated heparin and low molecular weight heparins binds to the Spike (S1) protein receptor binding domain, inducing distinct conformational change and we have further explored the structural features of heparin with regard to these interactions. In this article, previous research is expanded to now include a broader range of GAG family members, including heparan sulphate. This research demonstrates that GAGs, other than those of heparin (or its derivatives), can also interact with the SARS-CoV-2 Spike S1 receptor binding domain and induce distinct conformational changes within this region. These findings pave the way for future research into next-generation, tailor-made, GAG-based antiviral agents, against SARS-CoV-2 and other members of the Coronaviridae.

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pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

Protein and Peptide Letters ◽

10.2174/0929866526666190617092944 ◽

2019 ◽

Vol 26 (10) ◽

pp. 743-750 ◽

Cited By ~ 1

Author(s):

Remya Radha ◽

Sathyanarayana N. Gummadi

Keyword(s):

Vibrio Cholerae ◽

Conformational Changes ◽

Kinetic Studies ◽

Structural Features ◽

Structure Stability ◽

Kcal Mole ◽

Scanning Calorimetry ◽

Wide Range ◽

Ph Dependent ◽

Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

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Entropic Inhibition: How the Activity of a AAA+ Machine Is Modulated by Its Substrate-Binding Domain

ACS Chemical Biology ◽

10.1021/acschembio.1c00156 ◽

2021 ◽

Author(s):

Marija Iljina ◽

Hisham Mazal ◽

Pierre Goloubinoff ◽

Inbal Riven ◽

Gilad Haran

Keyword(s):

Substrate Binding ◽

Binding Domain ◽

Substrate Binding Domain

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Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships

Bioinformatics and Biology Insights ◽

10.4137/bbi.s9902 ◽

2012 ◽

Vol 6 ◽

pp. BBI.S9902 ◽

Cited By ~ 3

Author(s):

Divya P. Syamaladevi ◽

Margaret S Sunitha ◽

S. Kalaimathy ◽

Chandrashekar C. Reddy ◽

Mohammed Iftekhar ◽

...

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Homo Sapiens ◽

Relevant Literature ◽

Myosin Ii ◽

Coiled Coil ◽

Structural Features ◽

Model Organisms ◽

Congenital Diseases ◽

C Elegans

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms ( Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome .

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p190 RhoGAP, the major RasGAP-associated protein, binds GTP directly.

Molecular and Cellular Biology ◽

10.1128/mcb.14.11.7173 ◽

1994 ◽

Vol 14 (11) ◽

pp. 7173-7181 ◽

Cited By ~ 40

Author(s):

R Foster ◽

K Q Hu ◽

D A Shaywitz ◽

J Settleman

Keyword(s):

Structural Features ◽

Cellular Protein ◽

Small Gtpases ◽

Sequence Motifs ◽

Amino Terminal ◽

Terminal Domain ◽

Gtp Binding ◽

Guanine Nucleotide Binding ◽

Complex Forms ◽

Carboxy Terminal

In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.

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