Identification of endogenous ligands bound to bacterially expressed human and<i>E. coli</i>dihydrofolate reductase by 2D NMR

Bhabha, Gira; Tuttle, Lisa M.; Martinez-Yamout, Maria A.; Wright, Peter E.

doi:10.1016/j.febslet.2011.10.014

Cited by 14 publications

(19 citation statements)

References 19 publications

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“…Frozen cell pellets can be stored for several weeks at −20 °C. hDHFR was purified and the desired complex was prepared by refolding as previously described 37 .…”

Section: Methodsmentioning

confidence: 99%

“…Affinity purification was carried out using NiNTA resin (Qiagen), and protein was eluted with 50 mM Tris pH 8.0, 200 mM NaCl, 1mM folic acid, 250 mM imidazole. Fractions containing DHFR were further purified by reversed-phase HPLC, and refolded previously described 37 . DHFR from each species was divided into two aliquots; one was refolded with NADP + and FOL, while the other was refolded with NADP + and THF.…”

Section: Methodsmentioning

confidence: 99%

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Divergent evolution of protein conformational dynamics in dihydrofolate reductase

Bhabha

Ekiert

Jennewein

et al. 2013

Nat Struct Mol Biol

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161

204

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Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, E. coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics, following a pattern of divergent evolution that is tuned by the cellular environment.

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“…Frozen cell pellets can be stored for several weeks at −20 °C. hDHFR was purified and the desired complex was prepared by refolding as previously described 37 .…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Divergent evolution of protein conformational dynamics in dihydrofolate reductase

Bhabha

Ekiert

Jennewein

et al. 2013

Nat Struct Mol Biol

Self Cite

161

204

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“…Samples for assignments and 3 J coupling experiments were uniformly 13 C, 15 N-labeled and were purified as described previously. 25 NADPH and THF are light and air sensitive, and all samples containing these ligands were prepared in an argon-equilibrated glove box. The NMR buffer was thoroughly degassed under vacuum through a freeze-pump-thaw cycle and the protein was exchanged into NMR buffer [pH 7.6, 70 mM KPi, 25 mM KCl, 1 mM EDTA, 1 mM DTT, 10% D 2 O].…”

Section: Methodsmentioning

confidence: 99%

Side-Chain Conformational Heterogeneity of Intermediates in the Escherichia coli Dihydrofolate Reductase Catalytic Cycle

2013

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Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the χ1 rotamer populations of the aromatic and γ-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of 3JCγCO and 3JCγN coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond timescale. This large volume of 3J data has allowed us to fit new Karplus parameterizations for aromatic side chains, and to select the best available of previously determined parameters for Ile, Thr, and Val. The 3JCγCO and 3JCγN coupling constants are found to be extremely sensitive measures of side chain χ1 rotamers, and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand.

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“…In addition, this pathway does not exist in mammals; thus, the folate biosynthesis pathway has been considered a potential target during antibacterial compound development (31,32). In particular, folA is responsible for the reduction of dihydrofolate to tetrahydrofolate, which is crucial for protein and nucleic acid biosynthesis; folA is a well-studied target for the antibiotics trimethoprim and pyrimethamine (33)(34)(35)(36).…”

Section: Go Analysis Of the Unique Hits Of Each Amp-differentmentioning

confidence: 99%

Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays

Shah

Chen

et al. 2016

Molecular & Cellular Proteomics

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Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA-and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this Natural antimicrobial peptides (AMPs)1 are an evolutionarily conserved defense system of organisms against invading microorganisms. AMPs are effective against a wide range of microorganisms including bacteria, fungi, parasites, and some viruses (1). In general, AMPs are cationic peptides with fewer than 50 amino acid residues. To date, two main mechanisms of action have been identified for AMPs acting as antimicrobial agents (2): (1) membrane integrity disruption and (2) intracellular activity inhibition. Although the basic properties of all AMPs are similar, each AMP has a unique structure and microbial intracellular activity inhibition mechanism. For example, indolicidin inhibits DNA synthesis (3), whereas buforin II binds to DNA and RNA (4), mersacidin (a lantibiotic) binds to Lipid II and blocks peptidoglycan metabolism (5), tachyplesin I binds to the minor groove of DNA duplexes (6), microcin B17 inhibits DNA gyrase (thus influencing DNA replication) (7,8), microcin J25 recognizes the secondary channel of RNA polymerase (inhibiting transcription) (9), and pyrrhocoricin inhibits the biological function of the heat shock protein, DnaK (10). However, because no systematic study has been reported, the intracellular targets of numerous AMPs, such as b...

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Identification of endogenous ligands bound to bacterially expressed human andE. colidihydrofolate reductase by 2D NMR

Cited by 14 publications

References 19 publications

Divergent evolution of protein conformational dynamics in dihydrofolate reductase

Divergent evolution of protein conformational dynamics in dihydrofolate reductase

Side-Chain Conformational Heterogeneity of Intermediates in the Escherichia coli Dihydrofolate Reductase Catalytic Cycle

Systematic Analysis of Intracellular-targeting Antimicrobial Peptides, Bactenecin 7, Hybrid of Pleurocidin and Dermaseptin, Proline–Arginine-rich Peptide, and Lactoferricin B, by Using Escherichia coli Proteome Microarrays

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