Hemmingsen, Lars Bo Stegeager

Jancsó, Attila

Larsen, Flemming Hofmann

Roszahegyi, Livia

Szokolai, Hajnalka

Szunyogh, Daniel

Thulstrup, Peter Waaben

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetMetal ion interaction of an oligopeptide fragment representing      the regulatory metalbinding site of a CueR proteinJancsó, Attila; Szokolai, Hajnalka; Roszahegyi, Livia; Szunyogh, Daniel; Hemmingsen, LarsBo Stegeager; Thulstrup, Peter Waaben; Larsen, Flemming HofmannPublication date:2013Document versionEarly version, also known as pre-printCitation for published version (APA):Jancsó, A., Szokolai, H., Roszahegyi, L., Szunyogh, D., Hemmingsen, L. B. S., Thulstrup, P. W., & Larsen, F. H.(2013). Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of aCueR protein.Download date: 03. Feb. 2020Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of a CueR protein Attila Jancsó1,2, Hajnalka Szokolai1, Lívia Rózsahegyi1, Dániel M. Szunyogh1, Lars Hemmingsen3, Peter W. Thulstrup3, Flemming H. Larsen3 1MTA-SZTE Bioinorganic Chemistry Research Group, Hungary; 2Department of Inorganic and Analytical Chemistry, University of Szeged, Szeged, Hungary; 3Faculty of Life Sciences, University of Copenhagen, Copenhagen, Denmark E-mail: jancso@chem.u-szeged.hu Species distribution curves for the Cd2+:PP 0.5:1 system (cPP = 110–3 M). Red circles represent the recorded absorbances at l = 245 nm (cPP = 210–4 M, l = 0.5 cm). Introduction  Metalloregulatory proteins of the MerR family are transcriptional activators that sense/control the concentration of various metal ions inside bacteria.1 The Cu+ efflux regulator CueR, similarly to other MerR proteins, possesses a short multiple Cys-containing metal binding loop close to the C-terminus. CueR has a high selectivity for Cu+, Ag+ and Au+, but exhibits no transcriptional activity for the divalent ions Hg2+ and Zn2+.2 The two Cys-residues of the metal binding loop were shown to settle M+ ions into a linear coordination environment but other factors may also play a role in the recognition of cognate metal ions.2 Nevertheless, it is an interesting question whether the same sequence, when removed from the protein, shows a flexibility to adopt different coordination environments and may efficiently bind metal ions having preferences for larger coordination numbers.  Hg2+ - PP and Ag+ - PP systems  The significant downfield shift of the Cys CβH2 resonances, relative to those of the unbound PP ligand (data not shown), reflect a tight Hg2+ binding through the two distinct Cys-thiolates in a linear coordination fashion already at pH~2. In contrast, PP presumably coordinates Ag+ in a monodentate fashion below pH~7 and the second thiolate can be proposed to bind only above neutral pH. Aims  To address these questions a 12-mer peptide (PP), comprising the metal binding loop of V. cholerae CueR, has been synthesized and the Zn2+, Cd2+, Hg2+ and Ag+ binding of the ligand has been investigated by various techniques. Conclusions  The presented results demonstrate the higher coordination flexibility of the ligand, as compared to the metal binding site of native CueR. Most interestingly, preliminary studies with in the Ag+ - PP system suggest that the linear dicoordinate geometry proposed for the metal loaded form of the protein is retained only above neutral pH in the peptidic model. Schematic structure of the studied Ac-SCPGDQGSDCPI-NH2 (PP) peptide  Cd2+ - PP and Zn2+ - PP systems  LMCT bands emerging below pH ~ 6 on the UV-spectra recorded in the M2+:L 1:1 systems refer to the binding of both Cys-S– groups to the metal ions. 1H NMR spectra measured under similar conditions suggest the participation of Asp-carboxylate(s) in metal ion coordination, besides the Cys-residues, in the monomeric ML species. UV-spectra reveal a tetrahedral {4S} coordination mode in the ML2 complexes. 0.030.190.350.510.670.830.990204060801002.8 3.8 4.8 5.8 6.8 7.8 8.8 9.8 10.8A245 nm% CdIIpHCd2+CdHLCdLCdHL2CdL2Cd2LAcknowledgements  Financial support of the European Regional Development Fund (project No. HURO/1001/232/2.2.2. (METCAP) is acknowledged. AJ wishes to thank the financial support of the János Bolyai Research Grant from the Hungarian Academy of Sciences. Approximated pH-dependent distribution of monomeric species relative to the total fraction of such complexes in the Ag+:PP 1:1 system. (cPP = 110–3 M). 0.00.10.20.30.40.50.60.70.80.91.02.7 3.7 4.7 5.7 6.7 7.7 8.7 9.7 10.7A245 nmpHCd2+:PP 1:1Cd2+:PP 0.5:1PP without metal ionA vs. pH profiles recorded in the Cd2+:PP system at l = 245 nm  (cPP = 210–4 M, l = 0.5 cm). Selected region of the 1H NMR spectra of PP recorded at pH ~ 7.5 as a function of the Zn2+:peptide ratio (cPP = 110–3 M). Selected region of the 1H NMR spectra of the Ag+:PP 1:1 system as a function of pH (cPP = 110–3 M). The filled and open arrows point to the bound and unbound Cys CbH2 signals, respectively. 4.4 3.2 2.4 2.0 1.6 d /ppm3.64.0 2.8 1.2pH10.08.88.07.46.05.14.13.62.0    ML   ML2 Zn2+ :         {2S–,COO –,H2O}       {4S–} Cd2+ :         {2S–,COO –,H2O}       {4S–} Hg2+ :            {2S–}                     – Ag+ :            {2S–}                     – Proposed coordination modes of the peptide within the monomeric and metal-bridged structures formed with various metal ions. References  1, Ma et. al., Chem. Rev., 109, 4644, 2009. 2, Changela et. al., Science, 301, 1383, 2003. 0204060801002.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5% AgI monopHAgH3LAgLAgHLAgH2L4.5 4.2 3.3 2.7 2.4 2.1 d /ppm3.63.9 3.0Zn2+ : PP1 : 10.5 : 10 : 12,10C CbH25,9D CbH2NHOOHCH3CO NHOSHNONHONHOOOHNHOONH2NHONHOOHNHOOOHNHOSHNONHOCH3CH3NH2

Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of a CueR protein

https://curis.ku.dk/portal/files/48680774/Poster_ICBIC16_2013.pdf

Metal ion interaction of an oligopeptide fragment representing the regulatory metal binding site of a CueR protein

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