Sequence-Dependent Peptide Binding Orientation by the Molecular Chaperone DnaK

Tapley, Tim L.; Cupp‐Vickery, Jill R.; Vickery, Larry E.

doi:10.1021/bi051145r

Cited by 27 publications

(25 citation statements)

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“…The finding that the preferred orientation for peptides binding to HscA is sequence-dependent agrees with our previous studies on DnaK (39). In that work, peptides containing proline in the central positions were found to bind in the opposite direction of peptides containing leucine residues, and modeling studies suggested that geometrical constraints of proline residues restrict the peptide conformation to one that favors the reverse-binding orientation (39).…”

Section: Sequence Determinants For Iscu Peptide Binding To Hscasupporting

confidence: 91%

“…This indicates that the NR peptides bind to HscA in the forward direction. This binding orientation is opposite that observed for the HscA-ELPPVKI peptide complexes (Figure 3; 30, 31) but is the same as observed for the NR peptide binding in the DnaK(SBD)-NRLLLTG crystal structure (27) and full-length DnaK in solution (39). The finding that the preferred orientation for peptides binding to HscA is sequence-dependent agrees with our previous studies on DnaK (39).…”

Section: Sequence Determinants For Iscu Peptide Binding To Hscasupporting

confidence: 87%

“…This binding orientation is opposite that observed for the HscA-ELPPVKI peptide complexes (Figure 3; 30, 31) but is the same as observed for the NR peptide binding in the DnaK(SBD)-NRLLLTG crystal structure (27) and full-length DnaK in solution (39). The finding that the preferred orientation for peptides binding to HscA is sequence-dependent agrees with our previous studies on DnaK (39). In that work, peptides containing proline in the central positions were found to bind in the opposite direction of peptides containing leucine residues, and modeling studies suggested that geometrical constraints of proline residues restrict the peptide conformation to one that favors the reverse-binding orientation (39).…”

Section: Sequence Determinants For Iscu Peptide Binding To Hscamentioning

confidence: 45%

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Structural Determinants of HscA Peptide-Binding Specificity

2006

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The Hsp70-class molecular chaperone HscA interacts specifically with a conserved (99)LPPVK(103) motif of the iron-sulfur cluster scaffold protein IscU. We used a cellulose-bound peptide array to perform single-site saturation substitution of peptide residues corresponding to Glu(98)-Ile(104) of IscU to determine positional amino acid requirements for recognition by HscA. Two mutant chaperone forms, HscA(F426A) with a DnaK-like arch structure and HscA(M433V) with a DnaK-like substrate-binding pocket, were also studied. Wild-type HscA and HscA(F426A) exhibited a strict preference for proline in the central peptide position (ELPPVKI), whereas HscA(M433V) bound a peptide containing a Pro-->Leu substitution at this location (ELPLVKI). Contributions of Phe(426) and Met(433) to HscA peptide specificity were further tested in solution using a fluorescence-based peptide-binding assay. Bimane-labeled HscA and HscA(F426A) bound ELPPVKI peptides with higher affinity than leucine-substituted peptides, whereas HscA(M433V) favored binding of ELPLVKI peptides. Fluorescence-binding studies were also carried out with derivatives of the peptide NRLLLTG, a model substrate for DnaK. HscA and HscA(F426A) bound NRLLLTG peptides weakly, whereas HscA(M433V) bound NRLLLTG peptides with higher affinity than IscU-derived peptides ELPPVKI and ELPLVKI. These results suggest that the specificity of HscA for the LPPVK recognition sequence is determined in part by steric obstruction of the hydrophobic binding pocket by Met(433) and that substitution with the Val(433) sidechain imparts a broader, more DnaK-like, substrate recognition pattern.

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Section: Sequence Determinants For Iscu Peptide Binding To Hscasupporting

confidence: 91%

Section: Sequence Determinants For Iscu Peptide Binding To Hscasupporting

confidence: 87%

Section: Sequence Determinants For Iscu Peptide Binding To Hscamentioning

confidence: 45%

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Structural Determinants of HscA Peptide-Binding Specificity

2006

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“…27,28 However, the ability of DnaK to bind peptides in both directions has been suggested previously following spectroscopic studies. 29 Both binding modes of the Api88 fragment appear to be stabilized by crystal packing interactions. Substitution of glutamine in position 10 by arginine neither weakened nor strengthened the DnaK binding, which also agrees with the MIC values for E. coli.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug-Resistant Gram-Negative Pathogens

Czihal¹,

Knappe²,

Fritsche³

et al. 2012

ACS Chem. Biol.

115

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The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.

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“…The MIC values of these Api137 analogs indicated that the N-terminal sequence tolerated substitutions without major effects on the antibacterial activity (Fig. 4A), although apidaecins and Api88 are presumed to inhibit DnaK by binding of residues 3 to 11 to the substrate binding domain of DnaK (19,35,36). Substitutions of C-terminal residues (Pro-11 to Leu-18) significantly reduced the antibacterial activity in most cases.…”

Section: Fig 1 Degradation Of Api88 In 25% Aqueous Mouse Serum (Top) mentioning

confidence: 98%

Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

Berthold

Czihal

Fritsche

et al. 2013

Antimicrob Agents Chemother

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dProline-rich antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their pharmaceutical potential in treating systemic bacterial infections. Besides the native peptides, several shortened, modified, or even artificial sequences were highly effective in different murine infection models. Most recently, we showed that the 18-residue-long peptide Api88, an optimized version of apidaecin 1b, was efficient in two different animal infection models using the pathogenic Escherichia coli strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal leucine residue. To improve its in vitro characteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 with Lornithine or L-homoarginine increased the serum stabilities by more than 20-fold (half-life, ϳ4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is N,N,N=,N=-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine and D-amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an E. coli strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient in vivo than Api88.

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Sequence-Dependent Peptide Binding Orientation by the Molecular Chaperone DnaK

Cited by 27 publications

References 27 publications

Structural Determinants of HscA Peptide-Binding Specificity

Structural Determinants of HscA Peptide-Binding Specificity

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug-Resistant Gram-Negative Pathogens

Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

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