The crystal structure of ribonuclease A in complex with thymidine‐3′‐monophosphate provides further insight into ligand binding

Doucet, Nicolas; Jayasundera, Thusitha B.; Simonović, Miljan; Loria, J. Patrick

doi:10.1002/prot.22754

Cited by 15 publications

(10 citation statements)

References 53 publications

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“…To determine the binding affinity of the 5′-AMP and d(ACACA) ligands to RNase 6, we calculated the dissociation constants ( K d ) by fitting compounded chemical shift as a function of ligand concentration for each of the residues displaying Δδ > 0.05 ppm using eq . By calculating a shared value for all affected residues exhibiting straight resonance displacements typical of a fast exchange regime (Figure S6), we obtained a measured K d = 154 ± 5 μM for 5′-AMP binding to RNase 6, similar to ITC values previously reported for RNase A with the same ligand (105 ± 1 and 124 ± 4 μM). These values suggest similar properties for 5′-AMP binding to the B 2 purine subsite of RNase 6 and RNase A.…”

Section: Resultssupporting

confidence: 73%

Insights into Structural and Dynamical Changes Experienced by Human RNase 6 upon Ligand Binding

et al. 2020

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Ribonuclease 6 (RNase 6) is one of eight catalytically active human pancreatic-type RNases that belong to a superfamily of rapidly evolving enzymes. Like some of its human homologues, RNase 6 exhibits host defense properties such as antiviral and antibacterial activities. Recently solved crystal structures of this enzyme in its nucleotide-free form show the conservation of the prototypical kidney-shaped fold preserved among vertebrate RNases, in addition to revealing the presence of a unique secondary active site. In this study, we determine the structural and conformational properties experienced by RNase 6 upon binding to substrate and product analogues. We present the first crystal structures of RNase 6 bound to a nucleotide ligand (adenosine 5′-monophosphate), in addition to RNase 6 bound to phosphate ions. While the enzyme preserves B2 subsite ligand preferences, our results show a lack of typical B2 subsite interactions normally observed in homologous ligand-bound RNases. A comparison of the dynamical properties of RNase 6 in its apo-, substrate-, and product-bound states highlight the unique dynamical properties experienced on time scales ranging from nano- to milliseconds. Overall, our results confirm the specific evolutionary adaptation of RNase 6 relative to its unique catalytic and biological activities.

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Section: Resultssupporting

confidence: 73%

Insights into Structural and Dynamical Changes Experienced by Human RNase 6 upon Ligand Binding

et al. 2020

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“…We note that RNases 2 and 3 are the only two naturally occurring human RNase subtypes that lack the His48-Thr82 hydrogen bonding interaction that modulates the dynamics of loop 1 and display a corresponding reduction in catalytic activity relative to RNase A and other highly active RNases 15 . The top ranked residues of IC5 (His48 and Thr82) were shown previously using NMR relaxation experiments to form a hydrogen-bonding network (Figure S2) essential for modulating the conformational exchange of loop 1, which is coupled to catalytic activity in the bovine RNase A 5, 6, 16, 17 . Mutation of either residue was shown to result in the loss of conformational exchange 5, 6, 18 and a corresponding reduction in the catalytic turnover 6 .…”

Section: Resultsmentioning

confidence: 81%

Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily

Narayanan

Gagné

Reynolds

et al. 2017

Sci Rep

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In this work, we applied the sequence-based statistical coupling analysis approach to characterize conserved amino acid networks important for biochemical function in the pancreatic-type ribonuclease (ptRNase) superfamily. This superfamily-wide analysis indicates a decomposition of the RNase tertiary structure into spatially distributed yet physically connected networks of co-evolving amino acids, termed sectors. Comparison of this statistics-based description with new NMR experiments data shows that discrete amino acid networks, termed sectors, control the tuning of distinct functional properties in different enzyme homologs. Further, experimental characterization of evolutionarily distant sequences reveals that sequence variation at sector positions can distinguish homologs with a conserved dynamic pattern and optimal catalytic activity from those with altered dynamics and diminished catalytic activities. Taken together, these results provide important insights into the mechanistic design of the ptRNase superfamily, and presents a structural basis for evolutionary tuning of function in functionally diverse enzyme homologs.

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“…Protein/peptide interactions with metals and ions Protein/peptide interactions with nucleic acids Lipid/membrane interactions …”

Section: Introductionmentioning

confidence: 99%

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

Ghai

Falconer

Collins

2011

J of Molecular Recognition

167

108

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Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.

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The crystal structure of ribonuclease A in complex with thymidine‐3′‐monophosphate provides further insight into ligand binding

Cited by 15 publications

References 53 publications

Insights into Structural and Dynamical Changes Experienced by Human RNase 6 upon Ligand Binding

Insights into Structural and Dynamical Changes Experienced by Human RNase 6 upon Ligand Binding

Conserved amino acid networks modulate discrete functional properties in an enzyme superfamily

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

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