Investigating Protein−Ligand Interactions with a Mutant FKBP Possessing a Designed Specificity Pocket

Wu, Yang; Rozamus, Leonard W.; Narula, Surinder S.; Rollins, Carl T.; Yuan, Ruth W.; Andrade, Lawrence; Ram, Mary K.; Phillips, Thomas B.; Schravendijk, Marie Rose van; Dalgarno, David C.; Clackson, Tim; Holt, Dennis A.

doi:10.1021/jm9904396

Cited by 86 publications

(89 citation statements)

References 27 publications

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“…F M also retains the ability to bind with high affinity (K d Ϸ 1 nM; data not shown) to the ''bumped'' fully synthetic ligands we previously designed for the F36V mutant (11,12). Because these ligands bind minimally to the endogenous wild-type FKBP (11,12) they are preferable for intracellular applications.…”

Section: Fm Self-interacts In a Mammalianmentioning

confidence: 93%

“…F M ligands AP21998 and AP22542 were prepared by using strategies analogous to those previously reported (10)(11)(12). Details of the syntheses will be described elsewhere and are available on request.…”

Section: Methodsmentioning

confidence: 99%

“…Wild-type FKBP and F M were expressed in Escherichia coli BL21(DE3) as untagged proteins from vector pET20b, essentially as described for F V (11). Wild-type FKBP was purified as described (12). F M was purified similarly, except that cell paste was lysed by using a French press (Aminco); the S100 column was equilibrated with 20 mM potassium phosphate (KP i ) buffer, pH 6.5, containing 200 mM NaCl, 2 mM EDTA, 5 mM DTT, and 0.02% NaN 3 (S100 buffer); and the S100 pool was dialyzed against 20 mM Tris⅐HCl, pH 7.4͞5 mM DTT͞1 mM EDTA and then passed over MacroPrep High Q resin (Bio-Rad) equilibrated in the same buffer.…”

Section: Methodsmentioning

confidence: 99%

“…Because these ligands bind minimally to the endogenous wild-type FKBP (11,12) they are preferable for intracellular applications. To test the ability of these ligands to antagonize transcription, we first modified them to improve their cellular permeance by 1-2 orders of magnitude (not shown).…”

Section: Fm Self-interacts In a Mammalianmentioning

confidence: 99%

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A ligand-reversible dimerization system for controlling protein–protein interactions

Rollins

Rivera

Woolfson

et al. 2000

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

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178

140

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Chemically induced dimerization provides a general way to gain control over intracellular processes. Typically, FK506-binding protein (FKBP) domains are fused to a signaling domain of interest, allowing crosslinking to be initiated by addition of a bivalent FKBP ligand. In the course of protein engineering studies on human FKBP, we discovered that a single point mutation in the ligandbinding site (Phe-36 3 Met) converts the normally monomeric protein into a ligand-reversible dimer. Two-hybrid, gel filtration, analytical ultracentrifugation, and x-ray crystallographic studies show that the mutant (FM) forms discrete homodimers with micromolar affinity that can be completely dissociated within minutes by addition of monomeric synthetic ligands. These unexpected properties form the basis for a ''reverse dimerization'' regulatory system involving F M fusion proteins, in which association is the ground state and addition of ligand abolishes interactions. We have used this strategy to rapidly and reversibly aggregate fusion proteins in different cellular compartments, and to provide an off switch for transcription. Reiterated FM domains should be generally useful as conditional aggregation domains (CADs) to control intracellular events where rapid, reversible dissolution of interactions is required. Our results also suggest that dimerization is a latent property of the FKBP fold: the crystal structure reveals a remarkably complementary interaction between the monomer binding sites, with only subtle changes in side-chain disposition accounting for the dramatic change in quaternary structure.

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Section: Fm Self-interacts In a Mammalianmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Fm Self-interacts In a Mammalianmentioning

confidence: 99%

See 2 more Smart Citations

A ligand-reversible dimerization system for controlling protein–protein interactions

Rollins

Rivera

Woolfson

et al. 2000

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

Self Cite

178

140

View full text Add to dashboard Cite

show abstract

“…The same dimerizer can be used for many purposes since its action depends on the constructed target (wild protein with binding domain of dimerizer). For increasing selectivity it is proposed to design the anchor part for the mutant binding domain to exclude the capability of dimerizer binding to normal cell proteins containing wild-type domain [125,126].…”

Section: Dimerizersmentioning

confidence: 99%

Protein‐protein interactions as a target for drugs in proteomics

et al. 2003

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Protein-protein interactions play a central role in numerous processes in the cell and are one of the main fields of functional proteomics. This review highlights the methods of bioinformatics and functional proteomics of protein-protein interaction investigation. The structures and properties of contact surfaces, forces involved in protein-protein interactions, kinetic and thermodynamic parameters of these reactions were considered. The properties of protein contact surfaces depend on their functions. The contact surfaces of permanent complexes resemble domain contacts or the protein core and it is reasonable to consider such complex formation as a continuation of protein folding. Characteristics of contact surfaces of temporary protein complexes share some similarities with active sites of enzymes. The contact surfaces of the temporary protein complexes have unique structure and properties and they are more conservative in comparison with active site of enzymes. So they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations were undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or, on the contrary, to induce protein dimerization.

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Peptidyl‐prolylcis/transIsomerases

Fischer

2008

Protein Science Encyclopedia

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The stereospecificity of peptidyl prolyl cis/trans isomerases (PPIases) was studied using tetrapeptide substrate analogs in which one amino acid residue was replaced by the cognate D-amino acid in various positions of the peptide chain. Reversed stereocenters around proline markedly increased the rate of the spontaneous trans to cis isomerization of the prolyl bond whereas cis to trans isomerizations were less sensitive. PPIases like human cyclophilin18, human FKBP12, Escherichia coli parvulin10 and the PPIase domain of E. coli trigger factor exhibited stereoselectivity demanding at the P I to P P P position of the substrate chain. The discriminating factor for stereoselectiv-ity was the lack of formation of the Michaelis complexes of the diastereomeric substrates. However, D-alanine at the P I position preserved considerable affinity to the active site, and largely prevented activation of the catalytic machinery for all PPIases investigated. z 1998 Federation of European Biochemical Societies.

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Investigating Protein−Ligand Interactions with a Mutant FKBP Possessing a Designed Specificity Pocket

Cited by 86 publications

References 27 publications

A ligand-reversible dimerization system for controlling protein–protein interactions

A ligand-reversible dimerization system for controlling protein–protein interactions

Protein‐protein interactions as a target for drugs in proteomics

Peptidyl‐prolylcis/transIsomerases

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