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Probing the pH-dependent structural features of -KTx_12.1, a potassium channel blocker from the scorpion Tityus serrulatus

Sérgio Oyama, Jr.^1,7, Primo Pristovek^2,7, Lorella Franzoni^3,7, Thelma A. Pertinhez¹, Eugenia Schininá⁴, Christian Lücke^5,8, Heinz Rüterjans⁵, Eliane Candiani Arantes⁶ and Alberto Spisni^1,3

¹ Center for Brazilian Molecular Structural Biology (CeBiME)—Brazilian Synchrotron Light Laboratory (LNLS), 13084-971 Campinas/SP, Brazil² National Institute of Chemistry, 1001 Ljubljana, Slovenia³ Department of Experimental Medicine, Section of Chemistry and Structural Biochemistry, University of Parma, 43100 Parma, Italy⁴ Department of Biochemical Sciences, University of Roma, 00133 Roma, Italy⁵ Institute of Biophysical Chemistry, J.W. Goethe University of Frankfurt, 60439 Frankfurt, Germany⁶ Department of Physics and Chemistry, University of São Paulo, 14040-903 Ribeirão Preto, Brazil

(RECEIVED September 17, 2004; FINAL REVISION December 23, 2004; ACCEPTED December 23, 2004)

Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin -KTx_12.1 interacts with various K⁺ channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied -KTx_12.1 using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an -helix and a triple-stranded -sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK_a of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin’s coherent motions. In conclusion, His28, with its low pK_a value, provides -KTx_12.1 with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins’ functionality.

Keywords: molecular dynamics; nuclear magnetic resonance; potassium channel; scorpion toxin; essential dynamics analysis

Abbreviations: BuTX, butantoxin • CVFF, consistent valence force field • DQF-COSY, double quantum filtered correlated spectroscopy • ED, essential dynamics • ESI, electron spray ionization • LCMS, liquid chromatography mass spectrometry • MALDI-TOF, matrix-assisted laser desorption ionization–time of flight • MD, molecular dynamics • NOE, nuclear Over-hauser effect • NOESY, nuclear Overhauser and exchange spectroscopy • PAGE, polyacrylamide gel electrophoresis • RMSD, root mean square deviation • RMSF, root mean square fluctuation • TFA, trifluoroacetic acid • TFE, trifluoroethanol • TOCSY, total correlation spectroscopy

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041131205.

Reprint requests to: Alberto Spisni, Department of Experimental Medicine, Section of Chemistry and Structural Biochemistry, University of Parma, Via Volturno, 39, 43100 Parma, Italy; e-mail: aspin{at}unipr.it; fax: +39-0521-903802.

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