Structural studies of a hyperthermophilic thymidylate kinase enzyme reveal conformational substates along the reaction coordinate

Biswas, Arpita; Shukla, Arpit; Chaudhary, Santosh Kumar; Santhosh, R.; Jeyakanthan, Jeyaraman; Sekar, K.

doi:10.1111/febs.14140

Cited by 12 publications

(7 citation statements)

References 47 publications

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“…Thus, the binding of both of the substrates at the active site would be random in sequence and will follow a random bi-bi mechanism for substrate binding. A similar mode of substrate binding was also observed in a close homologue, A. aeolicus TMPK (Biswas, Shukla, Chaudhary et al, 2017). This is further reinforced by studies on mouse TMPK, adenylate kinase and guanylate kinase (Cheng & Prusoff, 1973;Sheng et al, 1999;Li et al, 1996).…”

Section: Insight Into the Mode Of Substrate Binding From The Crystal supporting

confidence: 64%

“…Thus, Arg91 acts as a hub for suboptimal paths that connect ATP and TMP at the active site. Biochemical studies of TMPK from other organisms have shown that mutation of the residues (Arg91, Asp12 or Tyr99) leads to a loss of enzyme activity (Biswas, Shukla, Chaudhary et al, 2017;. The agreement between the network analysis and the experimental results from earlier studies motivated us to explore the roles of the residues found in the suboptimal paths between ATP and TMP.…”

Section: Trajectory and Network Analysis Of The Tttmpk-atp-mg 2+ -Tmpmentioning

confidence: 77%

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Structural and functional roles of dynamically correlated residues in thymidylate kinase

Chaudhary

Jeyakanthan

Sekar

2018

Acta Crystallogr D Struct Biol

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Thymidylate kinase is an important enzyme in DNA synthesis. It catalyzes the conversion of thymidine monophosphate to thymidine diphosphate, with ATP as the preferred phosphoryl donor, in the presence of Mg. In this study, the dynamics of the active site and the communication paths between the substrates, ATP and TMP, are reported for thymidylate kinase from Thermus thermophilus. Conformational changes upon ligand binding and the path for communication between the substrates and the protein are important in understanding the catalytic mechanism of the enzyme. High-resolution X-ray crystal structures of thymidylate kinase in apo and ligand-bound states were solved. This is the first report of structures of binary and ternary complexes of thymidylate kinase with its natural substrates ATP and ATP-TMP, respectively. Distinct conformations of the active-site residues, the P-loop and the LID region observed in the apo and ligand-bound structures revealed that their concerted motion is required for the binding and proper positioning of the substrate TMP. Structural analyses provide an insight into the mode of substrate binding at the active site. The residues involved in communication between the substrates were identified through network analysis using molecular-dynamics simulations. The residues identified showed high sequence conservation across species. Biochemical analyses show that mutations of these residues either resulted in a loss of activity or affected the thermal stability of the protein. Further, molecular-dynamics analyses of mutants suggest that the proper positioning of TMP is important for catalysis. These data also provide an insight into the phosphoryl-transfer mechanism.

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Section: Insight Into the Mode Of Substrate Binding From The Crystal supporting

confidence: 64%

Section: Trajectory and Network Analysis Of The Tttmpk-atp-mg 2+ -Tmpmentioning

confidence: 77%

Structural and functional roles of dynamically correlated residues in thymidylate kinase

Chaudhary

Jeyakanthan

Sekar

2018

Acta Crystallogr D Struct Biol

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“…Investigation and resolution of residue interaction network (RIN) is imperative for the understanding protein structure-function relationships (Amitai et al, 2004 ; Del et al, 2006 ; Vishveshwara et al, 2009 ). Recently, RIN analysis has been successfully applied to investigate mutation effects, protein folding, domain-domain communication, and catalytic activity (Dokholyan et al, 2002 ; Swintkruse, 2004 ; Del et al, 2006 ; Soundararajan et al, 2010 ; Boehr et al, 2013 ; Scaini et al, 2014 ; Biswas et al, 2017 ). Based on the interaction mode analysis, we found that 1,5-InsP 8 lost some interactions and formed novel interactions with the jasmonate receptor complex in different systems.…”

Section: Resultsmentioning

confidence: 99%

The Binding Mechanism Between Inositol Phosphate (InsP) and the Jasmonate Receptor Complex: A Computational Study

Cui

Yao

2018

Front. Plant Sci.

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Jasmonates are critical plant hormones, mediating stress response in plants and regulating plant growth and development. The jasmonate receptor is a multi-component complex, composed of Arabidopsis SKP-LIKE PROTEIN1 (ASK1), CORONATINE INSENSITIVE 1 (COI1), inositol phosphate (InsP), and jasmonate ZIM-domain protein (JAZ). COI1 acts as multi-component signaling hub that binds with each component. InsP is suggested to play important roles in the hormone perception. How InsP binds with COI1 and the structural changes in COI1 upon binding with InsP, JA-Ile, and JAZ are not well understood. In this study, we integrated multiple computational methods, such as molecular docking, molecular dynamics simulations, residue interaction network analysis and binding free energy calculation, to explore the effect of InsP on the dynamic behavior of COI1 and the recognition mechanism of each component of the jasmonate receptor complex. We found that upon binding with InsP, JA-Ile, and JAZ1, the structure of COI1 becomes more compact. The binding of InsP with COI1 stabilizes the conformation of COI1 and promotes the binding between JA-Ile or JAZ1 and COI1. Analysis of the network parameters led to the identification of some hub nodes in this network, including Met88, His118, Arg120, Arg121, Arg346, Tyr382, Arg409, Trp467, and Lys492. The structural and dynamic details will be helpful for understanding the recognition mechanism of each component and the discovery and design of novel jasmonate signaling pathway modulators.

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“…Other significant perturbations to urinary metabolites appear to involve nucleotides, specifically increased monophosphate nucleotides (AMP, UMP, IMP) and decreased diphosphate nucleotides (GDP, CDP) in RSV samples. Monophosphate nucleotides are converted to diphosphate nucleotides by nucleoside-monophosphate kinases (NMPK) [30], while the opposite conversion from diphosphate to monophosphate nucleotides is mediated by nucleoside pyrophosphatase/phosphodiesterases (NPPs) [31]. Hence, accumulation of monophosphate nucleotides and decrease in diphosphate nucleotides may be due to either decreased NMPK activity, and/or increased NPP activity in urine-associated tissues such as the kidney or urinary tract.…”

Section: Discussionmentioning

confidence: 99%

Combined Plasma and Urinary Metabolomics Uncover Metabolic Perturbations Associated with Severe Respiratory Syncytial Viral Infection and Future Development of Asthma in Infant Patients

et al. 2022

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A large percentage of infants develop viral bronchiolitis needing medical intervention and often develop further airway disease such as asthma. To characterize metabolic perturbations in acute respiratory syncytial viral (RSV) bronchiolitis, we compared metabolomic profiles of moderate and severe RSV patients versus sedation controls. RSV patients were classified as moderate or severe based on the need for invasive mechanical ventilation. Whole blood and urine samples were collected at two time points (baseline and 72 h). Plasma and urinary metabolites were extracted in cold methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), and data from the two biofluids were combined for multivariate data analysis. Metabolite profiles were clustered according to severity, characterized by unique metabolic changes in both plasma and urine. Plasma metabolites that correlated with severity included intermediates in the sialic acid biosynthesis, while urinary metabolites included citrate as well as multiple nucleotides. Furthermore, metabolomic profiles were predictive of future development of asthma, with urinary metabolites exhibiting higher predictive power than plasma. These metabolites may offer unique insights into the pathology of RSV bronchiolitis and may be useful in identifying patients at risk for developing asthma.

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Structural studies of a hyperthermophilic thymidylate kinase enzyme reveal conformational substates along the reaction coordinate

Abstract: Structural data are available in the PDB under the accession numbers 2PBR, 4S2E, 5H5B, 5XAI, 4S35, 5XB2, 5H56, 5XB3, 5H5K, 5XB5, and 5XBH.

Cited by 12 publications

References 47 publications

Structural and functional roles of dynamically correlated residues in thymidylate kinase

Structural and functional roles of dynamically correlated residues in thymidylate kinase

The Binding Mechanism Between Inositol Phosphate (InsP) and the Jasmonate Receptor Complex: A Computational Study

Combined Plasma and Urinary Metabolomics Uncover Metabolic Perturbations Associated with Severe Respiratory Syncytial Viral Infection and Future Development of Asthma in Infant Patients

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