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1.
Acta Pharm Sin B ; 12(10): 3924-3933, 2022 Oct.
Article in English | MEDLINE | ID: mdl-35702321

ABSTRACT

RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3Cpro) of Enterovirus 71, the causative agent of hand, foot and mouth disease in humans, altering the substrate binding region. Further, we re-purpose these hits towards the main protease (Mpro) of SARS-CoV-2 which shares the 3C-like fold and a similar active site. The hit fragments covalently link to the catalytic cysteine of Mpro to inhibit its activity. We demonstrate that targeting the active site cysteine of Mpro can have profound allosteric effects, distorting secondary structures to disrupt the active dimeric unit.

2.
J Biol Chem ; 297(5): 101317, 2021 11.
Article in English | MEDLINE | ID: mdl-34678313

ABSTRACT

Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , Multienzyme Complexes/chemistry , Phosphoglucomutase/chemistry , Phosphorus-Oxygen Lyases/chemistry , Protein Multimerization , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Crystallography, X-Ray , Multienzyme Complexes/genetics , Phosphoglucomutase/genetics , Phosphorus-Oxygen Lyases/genetics , Protein Domains , Protein Structure, Quaternary
3.
Nat Chem Biol ; 17(12): 1296-1304, 2021 12.
Article in English | MEDLINE | ID: mdl-34556858

ABSTRACT

Toxin-antitoxin (TA) systems are a large family of genes implicated in the regulation of bacterial growth and its arrest in response to attacks. These systems encode nonsecreted toxins and antitoxins that specifically pair, even when present in several paralogous copies per genome. Salmonella enterica serovar Typhimurium contains three paralogous TacAT systems that block bacterial translation. We determined the crystal structures of the three TacAT complexes to understand the structural basis of specific TA neutralization and the evolution of such specific pairing. In the present study, we show that alteration of a discrete structural add-on element on the toxin drives specific recognition by their cognate antitoxin underpinning insulation of the three pairs. Similar to other TA families, the region supporting TA-specific pairing is key to neutralization. Our work reveals that additional TA interfaces beside the main neutralization interface increase the safe space for evolution of pairing specificity.


Subject(s)
Antitoxins/chemistry , Bacterial Toxins/chemistry , Recombinant Proteins/chemistry , Amino Acid Sequence , Antitoxins/genetics , Bacteria , Crystallization , Escherichia coli/genetics , Models, Molecular , Protein Binding , Protein Conformation , Recombinant Proteins/genetics , Toxin-Antitoxin Systems
4.
J Biol Chem ; 296: 100384, 2021.
Article in English | MEDLINE | ID: mdl-33556370

ABSTRACT

UTP-glucose-1-phosphate uridylyltransferases are enzymes that produce UDP-glucose from UTP and glucose-1-phosphate. In Bacillus subtilis 168, UDP-glucose is required for the decoration of wall teichoic acid (WTA) with glucose residues and the formation of glucolipids. The B. subtilis UGPase GtaB is essential for UDP-glucose production under standard aerobic growth conditions, and gtaB mutants display severe growth and morphological defects. However, bioinformatics predictions indicate that two other UTP-glucose-1-phosphate uridylyltransferases are present in B. subtilis. Here, we investigated the function of one of them named YngB. The crystal structure of YngB revealed that the protein has the typical fold and all necessary active site features of a functional UGPase. Furthermore, UGPase activity could be demonstrated in vitro using UTP and glucose-1-phosphate as substrates. Expression of YngB from a synthetic promoter in a B. subtilis gtaB mutant resulted in the reintroduction of glucose residues on WTA and production of glycolipids, demonstrating that the enzyme can function as UGPase in vivo. When WT and mutant B. subtilis strains were grown under anaerobic conditions, YngB-dependent glycolipid production and glucose decorations on WTA could be detected, revealing that YngB is expressed from its native promoter under anaerobic condition. Based on these findings, along with the structure of the operon containing yngB and the transcription factor thought to be required for its expression, we propose that besides WTA, potentially other cell wall components might be decorated with glucose residues during oxygen-limited growth condition.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Glycolipids/metabolism , Teichoic Acids/metabolism , UTP-Glucose-1-Phosphate Uridylyltransferase/metabolism , Anaerobiosis , Bacillus subtilis/growth & development , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Wall/metabolism , Crystallography, X-Ray/methods , Glycosylation , Promoter Regions, Genetic , Teichoic Acids/chemistry , UTP-Glucose-1-Phosphate Uridylyltransferase/chemistry , UTP-Glucose-1-Phosphate Uridylyltransferase/genetics
5.
Chembiochem ; 21(23): 3417-3422, 2020 12 01.
Article in English | MEDLINE | ID: mdl-32659037

ABSTRACT

Chemical probes that covalently modify cysteine residues in a protein-specific manner are valuable tools for biological investigations. Covalent fragments are increasingly implemented as probe starting points, but the complex relationship between fragment structure and binding kinetics makes covalent fragment optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data-driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing repertoire of methods for identifying covalent fragment hits by facilitating rapid multiparameter kinetic analysis of covalent structure-activity relationships through the simultaneous determination of Ki , kinact and intrinsic reactivity. By applying this approach to develop novel probes against electrophile-sensitive kinases, we showcase the utility of the platform in hit identification and highlight how multiparameter kinetic analysis enabled a successful fragment-merging strategy.


Subject(s)
Acrylamide/pharmacology , Cysteine/pharmacology , Fluorescent Dyes/pharmacology , Phosphotransferases/antagonists & inhibitors , Acrylamide/chemistry , Crystallography, X-Ray , Cysteine/chemistry , Fluorescent Dyes/chemistry , Humans , Kinetics , Models, Molecular , Molecular Structure , Phosphotransferases/metabolism , Structure-Activity Relationship , Thermodynamics
6.
Oncotarget ; 9(10): 9177-9198, 2018 Feb 06.
Article in English | MEDLINE | ID: mdl-29507682

ABSTRACT

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-specific function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant findings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was confirmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/stability of multiple chaperone client proteins could be perturbed by a deficient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identified. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our findings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP.

7.
Angew Chem Int Ed Engl ; 57(19): 5257-5261, 2018 05 04.
Article in English | MEDLINE | ID: mdl-29480525

ABSTRACT

Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine-reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-selective allosteric (type IV) kinase inhibitor whose novel mode-of-action could be exploited therapeutically.


Subject(s)
Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cysteine/pharmacology , Drug Discovery , High-Throughput Screening Assays , Ligands , Protein Kinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Cyclin-Dependent Kinase 2/metabolism , Cysteine/chemistry , Kinetics , Molecular Structure , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/chemical synthesis , Small Molecule Libraries/analysis , Small Molecule Libraries/chemical synthesis
8.
Front Mol Biosci ; 4: 68, 2017.
Article in English | MEDLINE | ID: mdl-29075633

ABSTRACT

Small glutamine-rich tetratricopeptide repeat-containing protein 2 (Sgt2) is a multi-module co-chaperone involved in several protein quality control pathways. The TPR domain of Sgt2 and several other proteins, including SGTA, Hop, and CHIP, is a highly conserved motif known to form transient complexes with molecular chaperones such as Hsp70 and Hsp90. In this work, we present the first high resolution crystal structures of Sgt2_TPR alone and in complex with a C-terminal peptide PTVEEVD from heat shock protein, Ssa1. Using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we demonstrate that Sgt2_TPR interacts with peptides corresponding to the C-termini of Ssa1, Hsc82, and Ybr137wp with similar binding modes and affinities.

9.
Int J Mol Sci ; 18(9)2017 Sep 07.
Article in English | MEDLINE | ID: mdl-28880248

ABSTRACT

The photochromic fluorescent protein Skylan-NS (Nonlinear Structured illumination variant mEos3.1H62L) is a reversibly photoswitchable fluorescent protein which has an unilluminated/ground state with an anionic and cis chromophore conformation and high fluorescence quantum yield. Photo-conversion with illumination at 515 nm generates a meta-stable intermediate with neutral trans-chromophore structure that has a 4 h lifetime. We present X-ray crystal structures of the cis (on) state at 1.9 Angstrom resolution and the trans (off) state at a limiting resolution of 1.55 Angstrom from serial femtosecond crystallography experiments conducted at SPring-8 Angstrom Compact Free Electron Laser (SACLA) at 7.0 keV and 10.5 keV, and at Linac Coherent Light Source (LCLS) at 9.5 keV. We present a comparison of the data reduction and structure determination statistics for the two facilities which differ in flux, beam characteristics and detector technologies. Furthermore, a comparison of droplet on demand, grease injection and Gas Dynamic Virtual Nozzle (GDVN) injection shows no significant differences in limiting resolution. The photoconversion of the on- to the off-state includes both internal and surface exposed protein structural changes, occurring in regions that lack crystal contacts in the orthorhombic crystal form.


Subject(s)
Crystallography, X-Ray/methods , Lasers , Luminescent Proteins/chemistry , Protein Conformation , Temperature
10.
J Clin Endocrinol Metab ; 101(8): 3144-54, 2016 08.
Article in English | MEDLINE | ID: mdl-27253664

ABSTRACT

CONTEXT: The pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. OBJECTIVE: This study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters. DESIGN AND SETTING: Half-life and protein-protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution. PATIENTS: Data were obtained from our cohort of pituitary adenoma patients and literature-reported cases. INTERVENTIONS: Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected. MAIN OUTCOME MEASURES: Half-life of wild-type and mutant AIP proteins and its correlation with clinical parameters. RESULTS: Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7-92.9 h), and those with short (median, 27 h; IQR, 21.6-28.7 h) or very short (median, 7.7 h; IQR, 5.6-10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1-CUL1-F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP. CONCLUSIONS: AIP is a stable protein, driven to ubiquitination by the SKP1-CUL1-F-box protein complex. Enhanced proteasomal degradation is a novel pathogenic mechanism for AIPmuts, with direct implications for the phenotype.


Subject(s)
Adenoma/genetics , Carcinogenesis , Intracellular Signaling Peptides and Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Pituitary Neoplasms/genetics , Proteolysis , Adenoma/metabolism , Adolescent , Adult , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cells, Cultured , Child , Cross-Sectional Studies , Female , HEK293 Cells , Humans , Male , Mutant Proteins/genetics , Pituitary Neoplasms/metabolism , Proteasome Endopeptidase Complex/metabolism , Young Adult
11.
Cell Rep ; 12(6): 1006-18, 2015 Aug 11.
Article in English | MEDLINE | ID: mdl-26235616

ABSTRACT

The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo.


Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-abl/metabolism , HEK293 Cells , HSP90 Heat-Shock Proteins/genetics , Humans , Immunoprecipitation , Models, Biological , Molecular Chaperones/genetics , Phosphorylation , Proto-Oncogene Proteins c-abl/genetics
12.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 5): 1197-206, 2015 May.
Article in English | MEDLINE | ID: mdl-25945584

ABSTRACT

Specific co-chaperone adaptors facilitate the recruitment of client proteins to the Hsp90 system. Tah1 binds the C-terminal conserved MEEVD motif of Hsp90, thus linking an eclectic set of client proteins to the R2TP complex for their assembly and regulation by Hsp90. Rather than the normal complement of seven α-helices seen in other tetratricopeptide repeat (TPR) domains, Tah1 unusually consists of the first five only. Consequently, the methionine of the MEEVD peptide remains exposed to solvent when bound by Tah1. In solution Tah1 appears to be predominantly monomeric, and recent structures have failed to explain how Tah1 appears to prevent the formation of mixed TPR domain-containing complexes such as Cpr6-(Hsp90)2-Tah1. To understand this further, the crystal structure of Tah1 in complex with the MEEVD peptide of Hsp90 was determined, which shows a helix swap involving the fifth α-helix between two adjacently bound Tah1 molecules. Dimerization of Tah1 restores the normal binding environment of the bound Hsp90 methionine residue by reconstituting a TPR binding site similar to that in seven-helix-containing TPR domain proteins. Dimerization also explains how other monomeric TPR-domain proteins are excluded from forming inappropriate mixed co-chaperone complexes.


Subject(s)
Cyclophilins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Multiprotein Complexes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Peptidyl-Prolyl Isomerase F , Cyclophilins/chemistry , HSP90 Heat-Shock Proteins/chemistry , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Chaperones/chemistry , Molecular Sequence Data , Multiprotein Complexes/chemistry , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology, Amino Acid
13.
PLoS One ; 7(12): e53339, 2012.
Article in English | MEDLINE | ID: mdl-23300914

ABSTRACT

Mutations of the aryl hydrocarbon receptor interacting protein (AIP) have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h) mutations, R304* (nonsense mutation), R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE) of TOMM20.


Subject(s)
Adenoma/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Pituitary Neoplasms/metabolism , Protein Binding/genetics , Adenoma/genetics , Genetic Predisposition to Disease , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Mutation , Pituitary Neoplasms/genetics , Protein Conformation
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