Distance fingerprinting: Pulsed electron–electron double resonance spectroscopy (PELDOR) is applied to the octameric membrane protein complex Wza of E. coli. The data yielded a detailed distance fingerprint of its periplasmic region that compares favorably to the crystal structure. These results provide the foundation to study conformation changes from interaction with partner proteins.

Archaea use a variety of small basic proteins to package their DNA. One of the most widespread and highly conserved is the Alba (Sso10b) protein. Alba interacts with both DNA and RNA in vitro, and we show in the present study that it binds more tightly to dsDNA (double-stranded DNA) than to either ssDNA (single-stranded DNA) or RNA. The Alba protein is dimeric in solution, and forms distinct ordered complexes with DNA that have been visualized by electron microscopy studies; these studies suggest that, on binding dsDNA, the protein forms extended helical protein fibres. An end-to-end association of consecutive Alba dimers is suggested by the presence of a dimer–dimer interface in crystal structures of Alba from several species, and by the strong conservation of the interface residues, centred on Arg59 and Phe60. In the present study we map perturbation of the polypeptide backbone of Alba upon binding to DNA and RNA by NMR, and demonstrate the central role of Phe60 in forming the dimer–dimer interface. Site-directed spin labelling and pulsed ESR are used to confirm that an end-to-end, dimer–dimer interaction forms in the presence of dsDNA.

The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.

Membrane proteins account for over one-fifth of the encoded proteins, but their crystallization is challenging, particularly for multiprotein complexes such as the polysaccharide export system of Escherichia coli. [1,2] One major component of this system is the translocation channel Wza, an octameric outermembrane protein of 320 kDa whose closed-state crystal structure has been recently determined.[3] Wza is thought to interact with different other proteins which, in part, reside in the inner membrane and cytosol, to form a periplasmspanning molecular machine. [1,2] The open-state structure of Wza is presumably stabilized during interaction with other proteins, and X-ray crystallography alone may not give a view of this dynamic complex. Pulsed electron-electron double resonance spectroscopy (PELDOR) is a powerful tool for measuring distances up to 80 . [4][5][6] Recently, the approach has been applied to study the maltose ATP binding cassette membrane protein complex and to quantify the internal motions during the catalytic cycle.[7] However, the approach has yet to be applied to large, highly symmetric integral membrane proteins such as Wza. This is an important gap, as many cellular processes involve highly symmetric membrane proteins. We show herein that PELDOR spectroscopy is wellsuited for the study of such a system, and a comparison of the PELDOR data with the crystal structure demonstrates the accuracy of the distance fingerprint. This fingerprint provides a convenient ruler by which to assess conformational changes.Two spin-labeled Wza species were made by expressing the single mutants G58C and Q335C of Wza and then reacting these mutants with the nitroxide MTSSL (methanethiosulfonate). Mass spectrometry was used to confirm the labeling. Since Wza is an eightfold-symmetric multimer, the eight labels (one in each monomer) give rise to four principle distances (Figure 1). PELDOR experiments were performed on these proteins solubilized in n-dodecyl-b-d-maltopyranoside (DDM) micelles , yielding modulated time traces and distance distributions (Figure 2 A,B). Two distinct distance peaks could be measured for each mutant, for Wza Q335C at 28.6 and 51 and for Wza G58C at 36.7 and 66 (Table 1). Taking the additional length of each label into account (ca. 9 ), these distances agree well with the C b ÀC b distances 1-2 and 1-3 (Figure 1 B) inferred from the crystal structure (Table 1).The purification of Wza is time-consuming, so we sought to investigate whether a soluble version of the protein could be produced that retained the same structural properties as full-length Wza. We expressed a truncated Wza 24-345 mutant (sWza; see the Supporting Information), which lacks the signal sequence and the C-terminal transmembrane domain D4. The structure of sWza was essentially identical to the corresponding domains from the full-length protein (see the Supporting Information). As the C-terminal transmembrane helices are unlikely to be involved in proteinprotein complex formation, sWza is a suitable system for further study...