Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

Lipscomb, Leigh Ann; Gassner, N.C.; Snow, S.; Eldridge, Aimee M.; Baase, W.A.; Drew, Devin L.; Matthews, B.W.

doi:10.1002/pro.5560070326

Cited by 55 publications

(47 citation statements)

References 26 publications

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“…Hence, it appears that the protein scaffold of CRBP-I is more compact, thus contributing to its increased structural stability compared with CRBP-II. A similar protein destabilization as a result of Met replacement has been reported for two Met-to-Leu mutants of T4 lysozyme, where each methionine residue is buried next to a Trp ring and at least one other aromatic moiety ( 44 ).…”

Section: H/supporting

confidence: 69%

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers

Franzoni

Cavazzini

Rossi

et al. 2010

Journal of Lipid Research

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Section: H/supporting

confidence: 69%

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers

Franzoni

Cavazzini

Rossi

et al. 2010

Journal of Lipid Research

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“…Because Met has a relatively hydrophobic side chain, which often substitutes for Leu in protein structures (24), it was not immediately apparent how unwanted ClpS-mediated degradation of proteins with N-terminal Met is avoided. We find that substrates with N-terminal Met bind ClpS with Ϸ1,000-fold reduced affinity when compared with authentic N-end rule residues.…”

Section: Discussionmentioning

confidence: 99%

Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

Roman-Hernandez¹,

Grant²,

Sauer³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The N-end rule is a conserved degradation pathway that relates the stability of a protein to its N-terminal amino acid. Here, we present crystal structures of ClpS, the bacterial N-end rule adaptor, alone and engaged with peptides containing N-terminal phenylalanine, leucine, and tryptophan. These structures, together with a previous structure of ClpS bound to an N-terminal tyrosine, illustrate the molecular basis of recognition of the complete set of primary N-end rule amino acids. In each case, the ␣-amino group and side chain of the N-terminal residue are the major determinants of recognition. The binding pocket for the N-end residue is preformed in the free adaptor, and only small adjustments are needed to accommodate N-end rule residues having substantially different sizes and shapes. M53A ClpS is known to mediate degradation of an expanded repertoire of substrates, including those with N-terminal valine or isoleucine. A structure of Met53A ClpS engaged with an N-end rule tryptophan reveals an essentially wild-type mechanism of recognition, indicating that the Met 53 side chain directly enforces specificity by clashing with and excluding ␤-branched side chains. Finally, experimental and structural data suggest mechanisms that make proteins with N-terminal methionine bind very poorly to ClpS, explaining why these high-abundance proteins are not degraded via the N-end rule pathway in the cell.AAAϩ ATPase adaptor ͉ clpAP protease ͉ degradation tag ͉ degron

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“…The entropic penalty resulting from constraining a methionine sidechain is larger than the corresponding cost for a leucine sidechain. The change of a buried leucine to a methionine has been shown experimentally to destabilize the protein with a total entropic and desolvation cost of 1.4 kcal/mol (Lipscomb et al, 1998).…”

Section: Docking Calculationsmentioning

confidence: 99%

6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: Exploitation of GSK-3 for treating leishmaniasis

Xingi

Smirlis

Myrianthopoulos

et al. 2009

International Journal for Parasitology

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Indirubins known to target mammalian cyclin-dependent kinases (CDKs) and glycogen synthase kinase (GSK-3) were tested for their antileishmanial activity. 6-Br-indirubin-3'-oxime (6-BIO), 6-Br-indirubin-3'acetoxime and 6-Br-5methylindirubin-3'oxime (5-Me-6-BIO) were the most potent inhibitors of L. donovani promastigote and amastigote growth (IC50 values ≤ 1.2 μM). Since the 6-Br substitution on the indirubin backbone greatly enhances the selectivity for mammalian GSK-3 over CDKs, we identified the leishmanial GSK-3 homologues, a short (LdGSK-3s) and a long one, focusing on LdGSK-3s which is closer to human GSK-3β for further studies. Kinase assays showed that 5-Me-6-BIO inhibited LdGSK-3s more potently than CRK3 (the CDK1 homologue in Leishmania), while 6-BIO was more selective for CRK3. Promastigotes treated with 5-Me-6-BIO accumulated in the S and G2/M cell-cycle phases and underwent apoptosis-like death.Interestingly, these phenotypes were completely reversed in parasites over-expressing LdGSK-3s.This finding strongly supports that LdGSK-3s is a) the intracellular target of 5-Me-6-BIO and b) involved in cell-cycle control and in pathways leading to apoptosis-like death. 6-BIO treatment induced a G2/M arrest, consistent with inhibition of CRK3, and apoptosis-like death. These effects were partially reversed in parasites over-expressing suggesting that in vivo 6-BIO may also target LdGSK-3s. Molecular docking of 5-Me-6-BIO in CRK3 and 6-BIO in human and LdGSK-3s active sites predict the existence of functional/structural differences that are sufficient to explain the observed difference in their affinity. In conclusion, LdGSK-3s is validated as a potential drug target in Leishmania and could be exploited for the development of selective indirubin-based leishmanicidals. AUTHOR AGREEMENT FORMManuscript title: "5-Me-6-BIO targeting the leishmanial GSK-3 short form affects cell-cycle progression and induces apoptosis-like death: exploitation of GSK-3 for treating leishmaniasis" Authors: Evangelia Xingi, Despina Smirlis , Vassilios Myrianthopoulos , Prokopios Magiatis, Karen M. Grant, Laurent Meijer, Emmanuel Mikros, Alexios-Leandros Skaltsounis and Ketty Soteriadou We, the undersigned, acknowledge that we have read the above manuscript and accept responsibility for its contents. We confirm this manuscript has not been published previously and if accepted in the International Journal for Parasitology will not be published elsewhere without the approval of the Editor-in-Chief. We also confirm there are no financial or other relationships that might lead to a conflict of interest.[If a financial or other relationship exists that might lead to a conflict of interest, please provide that information here.][If this letter is being submitted at revision and if there have been any changes in authors added to/deleted from the paper or if the order of author citation has changed since the original submission, please include a statement acknowledging those changes, before each author (including authors deleted fro...

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Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

Cited by 55 publications

References 26 publications

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers

Molecular basis of substrate selection by the N-end rule adaptor protein ClpS

6-Br-5methylindirubin-3′oxime (5-Me-6-BIO) targeting the leishmanial glycogen synthase kinase-3 (GSK-3) short form affects cell-cycle progression and induces apoptosis-like death: Exploitation of GSK-3 for treating leishmaniasis

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