Structure of the Month: January 2010 [see all]

The structural characterization of the redundant thioredoxin system of Sulfolobus solfataricus

Alessia Ruggiero¹, Mariorosario Masullo^2,3, Daniela Marasco^1,4, Maria Rosaria Ruocco³, Pasquale Grimaldi^2,3, Paolo Arcari³, Adriana Zagari^1,4 and Luigi Vitagliano^{1, *}

Living organisms have developed efficient biological mechanisms for scavenging reactive oxygen species produced by metabolism. The thioredoxin system, composed by thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH plays a crucial role in regulating the redox state of the cell and in protecting them against oxidative stress. Although this system is ubiquitous among organisms of all three domains of life, most of the functional and structural investigations have been hitherto focused on eukaryal and eubacteryal Trx systems. Limited information is indeed available for enzymes involved in the Trx system of archaea. We have recently undertaken the functional and structural characterization of the Trx system of the hyperthermophilic archeon Sulfolobus solfataricus. In previous studies we have shown that two distinct Trxs (SsTrxA2 and SsTrxA1) are substrates for the thioredoxin reductase SsTrxRB3 (1). On the other hand, two additional proteins SsTrxRB1 and SsTrxRB2, which share a significant sequence identity with members of the TrxR superfamily, are unable to reduce either SsTrxA2 or SsTrxA1 (1). In order to relate the functional properties of these enzymes to their three-dimensional structure and to elucidate at the molecular level the basis of Trx-TrxR molecular recognition process, we have also undertaken crystallographic investigations on the protein components of S. solfataricus Trx system. Using synchrotron and conventional (Rigaku 007HF generator) X-ray diffraction data we initially described the crystal structure of SsTrxRB3, which has provided clues on the determinants of its dual function (thioredoxin reductase and NADH oxidase) and thermostability (2). We have then undertaken a structural characterization of the two Trx SsTrxA2 and TrxA1.

For both proteins high resolution diffraction data were collected in-house at 100 K using a Rigaku MicroMax-007HF X-ray generator equipped with a Saturn 944 CCD detector. Crystals were flash cooled after the addition of 22% (v/v) glycerol to the crystallization buffer (3).

As expected from the significant sequence identity with other members of the family, SsTrxA2 adopts a typical thioredoxin fold characterized by a central five-stranded β-sheet with a ↑↑↑↓↑ topology surrounded by four α-helices (4). The analysis of SsTrxA2 redox center clearly indicates that the reactive disulfide bridge of the protein is in its oxidized state (Figure 1). The elucidation of SsTrxA2 structure reveals a peculiar dimeric organization (Figure 2). An interesting consequence of the specific monomer-monomer association in SsTrxA2 dimer is indeed the burying of the redox center of the protein. This hampers the access of the TrxR enzyme to the redox center of this protein in its dimeric state. Therefore, it is likely that the recognition mechanism of Trx by TrxR leads to the dissociation of the dimer.

The number of ion pairs detected in SsTrxA2 structure (three) is in line with that observed in the structures of Trx isolated from mesophilic organisms (4). This suggests that electrostatic interactions are not key determinants for the extraordinary thermal stability of the protein. On the other hand, the analysis of secondary structure elements indicates that SsTrxA2 presents shorter loops when compared with Trxs of mesophilic organisms. Since it is know that the shortening of loops and the extension of secondary structure elements increase the capability of the protein to protect its core from the solvent environment, we propose that SsTrxA2 is stabilized by these effects. This observation has been corroborated by chemical denaturation experiments conducted on this protein (Ruggiero et al, in preparation). 

The spectroscopic and crystallographic characterization of SsTrxA1 confirms the hypothesis that the shortening of the loop connecting secondary structure elements plays important role in the stabilization of thioredoxins (Ruggiero et al, in preparation).

This work was performed in the laboratory of Dr. Luigi Vitagliano of the Institute of Biostructures and Bioimaging (IBB) of the Italian National Research Council (CNR) and was published on Proteins 77,1004-1008, December 2009.

References

1. Grimaldi P, Ruocco MR, Lanzotti MA, Ruggiero A, Ruggiero I, Arcari P, Vitagliano L, Masullo M. Characterisation of the components of the thioredoxin system in the archaeon Sulfolobus solfataricus. Extremophiles 2008;12:553-562.
2. Ruggiero A, Masullo M, Ruocco MR, Grimaldi P, Lanzotti MA, Arcari P, Zagari A, Vitagliano L. Structure and stability of a thioredoxin reductase from Sulfolobus solfataricus: A thermostable protein with two functions. Biochim Biophys Acta 2009;1794:554-562.
3. Ruggiero A, Lanzotti MA, Ruocco MR, Grimaldi P, Marasco D, Arcari P, Masullo M, Zagari A, Vitagliano L. Crystallization and preliminary X-ray crystallographic analysis of two dimeric hyperthermostable thioredoxins isolated from Sulfolobus solfataricus. Acta Crystallogr F 2009;F65:604-7.
4. Ruggiero A, Masullo M, Marasco D, Ruocco MR, Grimaldi P, Arcari P, Zagari A, Vitagliano L. The dimeric structure of Sulfolobus solfataricus thioredoxin A2 and the basis of its thermostability. Proteins 2009; 77,1004-1008,.

Data collection parameters and data processing statistics