Dataset Information

System-wide identification of enzyme substrates by thermal analysis (SIESTA)

ABSTRACT: Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying the molecular components participating in these reactions on a cellular level. Here we developed a universal method called System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic post-translational modification of substrate proteins changes their thermal stability, and applies the concept of specificity to reveal potential substrates. We have already shown the applicability of SIESTA in substrate discovery for TXNRD1 and PARP10 enzymes. SIESTA successfully identified several known and novel substrate candidates for protein kinase B (AKT1). A number of putative substrates were confirmed by functional assays. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on protein stability, and facilitate drug discovery.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Epithelial Cell Of Cervix

DISEASE(S): Cervix Carcinoma

SUBMITTER: Amir Ata Saei

LAB HEAD: Roman Zubarev

PROVIDER: PXD014445 | Pride | 2021-01-11

REPOSITORIES: Pride

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Publications

System-wide identification and prioritization of enzyme substrates by thermal analysis.

Saei Amir Ata AA Beusch Christian M CM Sabatier Pierre P Wells Juan Astorga JA Gharibi Hassan H Meng Zhaowei Z Chernobrovkin Alexey A Rodin Sergey S Näreoja Katja K Thorsell Ann-Gerd AG Karlberg Tobias T Cheng Qing Q Lundström Susanna L SL Gaetani Massimiliano M Végvári Ákos Á Arnér Elias S J ESJ Schüler Herwig H Zubarev Roman A RA

Nature communications 20210226 1

Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change the ...[more]

PMID: 33637753

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Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.

Dataset Information

System-wide identification of enzyme substrates by thermal analysis (SIESTA)

Publications

System-wide identification and prioritization of enzyme substrates by thermal analysis.

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