1.Effect of diaminopropionic acid (Dap) on the biophysical properties of a modified synthetic channel-forming peptide.
Bukovnik U1, Sala-Rabanal M, Francis S, Frazier SJ, Schultz BD, Nichols CG, Tomich JM. Mol Pharm. 2013 Oct 7;10(10):3959-66. doi: 10.1021/mp4002377. Epub 2013 Sep 23.
Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions.
2.Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis.
Kobylarz MJ1, Grigg JC1, Liu Y1, Lee MS1, Heinrichs DE, Murphy ME1. Biochemistry. 2016 Feb 16;55(6):927-39. doi: 10.1021/acs.biochem.5b01045. Epub 2016 Feb 3.
Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate.
3.Ring size in cyclic endomorphin-2 analogs modulates receptor binding affinity and selectivity.
Piekielna J1, Kluczyk A, Gentilucci L, Cerlesi MC, Calo' G, Tomböly C, Łapiński K, Janecki T, Janecka A. Org Biomol Chem. 2015 Jun 7;13(21):6039-46. doi: 10.1039/c5ob00565e.
The study reports the solid-phase synthesis and biological evaluation of a series of new side chain-to-side chain cyclized opioid peptide analogs of the general structure Tyr-[D-Xaa-Phe-Phe-Asp]NH2, where Xaa = Lys (1), Orn (2), Dab (3), or Dap (4) (Dab = 2,4-diaminobutyric acid, Dap = 2,3-diaminopropionic acid), containing 17- to 14-membered rings. The influence of the ring size on binding to the MOP, DOP and KOP opioid receptors was studied. In general, the reduction of the size of the macrocyclic ring increased the selectivity for the MOP receptor. The cyclopeptide incorporating Xaa = Lys displayed subnanomolar MOP affinity but modest selectivity over the KOP receptor, while the analog with the Orn residue showed increased affinity and selectivity for MOP. The analog with Dab was a weak MOP agonist and did not bind to the other two opioid receptors. Finally, the peptide with Xaa = Dap was completely MOP receptor-selective with subnanomolar affinity.
4.A novel branched TAT(47-57) peptide for selective Ni(2+) introduction into the human fibrosarcoma cell nucleus.
Szyrwiel Ł1, Shimura M, Shirataki J, Matsuyama S, Matsunaga A, Setner B, Szczukowski Ł, Szewczuk Z, Yamauchi K, Malinka W, Chavatte L, Łobinski R. Metallomics. 2015 Jul;7(7):1155-62. doi: 10.1039/c5mt00021a.
A TAT47-57 peptide was modified on the N-terminus by elongation with a 2,3-diaminopropionic acid residue and then by coupling of two histidine residues on its N-atoms. This branched peptide could bind to Ni under physiological conditions as a 1 : 1 complex. We demonstrated that the complex was quantitatively taken up by human fibrosarcoma cells, in contrast to Ni(2+) ions. Ni localization (especially at the nuclei) was confirmed by imaging using both scanning X-ray fluorescence microscopy and Newport Green fluorescence. A competitive assay with Newport Green showed that the latter displaced the peptide ligand from the Ni-complex. Ni(2+) delivered as a complex with the designed peptide induced substantially more DNA damage than when introduced as a free ion. The availability of such a construct opens up the way to investigate the importance of the nucleus as a target for the cytotoxicity, genotoxicity or carcinogenicity of Ni(2+).