Structure of the Month: September 2008 [see all]

Anomalous diffraction used to confirm the metal ion in Tet repressor - tetracycline complexes

The antibiotic function of Tetracycline and its derivatives rely on blocking protein biosynthesis by binding to the prokaryotic ribosome [1]. Common problems in clinical application are resistance mechanisms due to the wide spread use of antibiotics. In gram-negative bacteria an efflux mechanism protects the ribosome by reducing the inner cell concentration of tetracycline. The expression of a specific membrane protein, TetA, is strictly controlled by the Tet repressor, TetR [2]. The repression is turned off by binding of the tetracycline to TetR (Fig. 1). The TetR/operator complex dissociates after tetracycline induces allosteric mechanisms upon binding to TetR [3,4]. This interaction is mediated by a divalent metal ion, physiologically a magnesium (2+)-ion [5]. This tetracycline-Mg(2+) complex is of general interest because it binds to the ribosome [1], induces TetR [4] and is the target which is recognized and transported by the membrane residing TetA protein [6].

We have analyzed binding of the biologically important divalent cations to tetracycline (Tc) and of the metal tetracycline complex to the Tet repressor [7]. Formation of the metal - tetracycline complex is strongly dependent on the divalent metal ion, the more abundant earth alkaline metal ions bind 10-100 times weaker than divalent transition metal ions. Subsequent binding of the complex to the Tet repressor (forming a ternary complex Tet repressor - metal - tetracycline) is basically independent of the type of metal ion. To study the coordination of the metal ion in the tetracycline binding site, we compared structures with different metal ions. The octahedral coordination and the ligands are well conserved in the Mg²⁺, Co²⁺ and Ni²⁺-complexes. The structure of the metal binding site is shown in Fig. 2. An anomalous map was calculated with phases from the refined structure and amplitudes based on the measured anomalous differences, see Fig 2. This map clearly shows the Co2+-ion with a peak of 40σ. For comparison, all three sulfur atoms (Met84, Met120 and Met177) and three solvent molecules identified as chlorides have 3-5σ. The replacement of Mg²⁺ by Co²⁺ is thus unambiguous.

This work was performed in the laboratory of Dr. Winfried Hinrichs at the University of Greifswald, in the Institute for Biochemistry. These results are part of an ongoing research project in collaboration with W. Hillen (Univ. Erlangen). The project is focused on structural biochemistry of new classes and mutations of the tetracycline repressor TetR.

Data collection details

Data collection details for the TetR/[Co Tc]⁺ complex. More data (to a resolution of 1.62 Å) were collected in the edges of the detector and used for refinement, but contributed only little.

References

[1] Pioletti M, Schlunzen F, Harms J, Zarivach R, Gluhmann M, Avila H, Bashan A, Bartels H, Auerbach T, Jacobi C, Hartsch T, Yonath A, Franceschi F (2001) EMBO J 20:1829-1839
[2] Hillen W, Berens C (1994) Annu Rev Microbiol 48:345-369
[3] Hinrichs W, Kisker C, Düvel M, Müller A, Tovar K, Hillen W, Saenger W (1994) Science 264:418-420
[4] Orth P, Schnappinger D, Hillen W, Saenger W, Hinrichs W (2000) Nat Struct Biol 7:215-219
[5] Orth P, Saenger W, Hinrichs W (1999) Biochemistry 38:191-198
[6] Yamaguchi A, Udagawa T, Sawai T (1990) J Biol Chem 265:4809-4813
[7] Palm GJ, Lederer T, Orth P, Saenger W, Takahashi M, Hillen W, Hinrichs W (2008) J Biol Inorg Chem, in press.