Edited by George M. Carman Fatty acid (FA) kinase produces acyl-phosphate for the synthesis of membrane phospholipids in Gram-positive bacterial pathogens. FA kinase consists of a kinase protein (FakA) that phosphorylates an FA substrate bound to a second module, an FA-binding protein (FakB). Staphylococcus aureus expresses two distinct, but related, FakBs with different FA selectivities. Here, we report the structures of FakB1 bound to four saturated FAs at 1.6-1.93 Å resolution. We observed that the different FA structures are accommodated within a slightly curved hydrophobic cavity whose length is governed by the conformation of an isoleucine side chain at the end of the tunnel. The hydrophobic tunnel in FakB1 prevents the binding of cis-unsaturated FAs, which are instead accommodated by the kinked tunnel within the FakB2 protein. The differences in the FakB interiors are not propagated to the proteins' surfaces, preserving the proteinprotein interactions with their three common partners, FakA, PlsX, and PlsY. Using cellular thermal shift analyses, we found that FakB1 binds FA in vivo, whereas a significant proportion of FakB2 does not. Incorporation of exogenous FA into phospholipid in ⌬fakB1 and ⌬fakB2 S. aureus knockout strains revealed that FakB1 does not efficiently activate unsaturated FAs. FakB2 preferred unsaturated FAs, but also allowed the incorporation of saturated FAs. These results are consistent with a model in which FakB1 primarily functions in the recycling of the saturated FAs produced by S. aureus metabolism, whereas FakB2 activates host-derived oleate, which S. aureus does not produce but is abundant at infection sites.

Streptococcus pneumoniae is responsible for the majority of pneumonia, motivating ongoing searches for insights into its physiology that could enable new treatments. S. pneumoniae responds to exogenous fatty acids by suppressing its de novo biosynthetic pathway and exclusively utilizing extracellular fatty acids for membrane phospholipid synthesis. The first step in exogenous fatty acid assimilation is phosphorylation by fatty acid kinase (FakA), whereas bound by a fatty acid-binding protein (FakB). Staphylococcus aureus has two binding proteins, whereas S. pneumoniae expresses three. The functions of these binding proteins were not clear. We determined the SpFakB1and SpFakB2-binding proteins were bioinformatically related to the two binding proteins of Staphylococcus aureus, and biochemical and X-ray crystallographic analysis showed that SpFakB1 selectively bound saturates, whereas SpFakB2 allows the activation of monounsaturates akin to their S. aureus counterparts. The distinct SpFakB3 enables the utilization of polyunsaturates. The SpFakB3 crystal structure in complex with linoleic acid reveals an expanded fatty acid-binding pocket within the hydrophobic interior of SpFakB3 that explains its ability to accommodate multiple cis double bonds. SpFakB3 also utilizes a different hydrogen bond network than other FakBs to anchor the fatty acid carbonyl and stabilize the protein. S. pneumoniae strain JMG1 (⌬fakB3) was deficient in incorporation of linoleate from human serum verifying the role of FakB3 in this process. Thus, the multiple FakBs of S. pneumoniae permit the utilization of the entire spectrum of mammalian fatty acid structures to construct its membrane.

Rubredoxins are small monomeric non-heme, mononuclear iron-sulfur cluster proteins found in prokaryotes [1] and in some eukaryotes. [2] Although a multitude of roles have been proposed, [3] the main functions of the rubredoxins remain uncertain. Rubredoxin from Pyrococcus furiosus (Pf) is one of the most thermostable proteins known, with a melting temperature of 200 8C, and it can resist denaturation over prolonged periods at temperatures of up to 95 8C. [4] The structure of the hyperthermophilic Pf rubredoxin does not contain an unusual number of hydrogen bonds but has a richly H-bonded N-terminus region involving residue Glu14, which is absent in mesophilic rubredoxins. It has been suggested that this may contribute to the high thermostability of Pf rubredoxin. [5] As a redox protein, rubredoxin is also an important model system for an understanding of electron transfer processes associated with catalysis. Iron-sulfur proteins occur widely, covering a wide range of redox potentials, and this variation may be attributable to variations in solvation, hydrogen bonding, and electrostatic interactions.The aim of the current work was to obtain high-resolution neutron crystallographic information from both oxidized and reduced forms of perdeuterated Pf rubredoxin by fully exploiting the power of neutrons to clearly image water molecules, hydronium ions, and the protonation of carboxylic and hydroxylic moieties. Neutron diffraction is the only method that can provide this level of detail. The D19 instrument at the Institut Laue-Langevin (ILL) is a monochromatic diffractometer that allows atomic resolution stud-ies of systems for which suitably large crystals are available. It complements the quasi-Laue instrument LADI-III, which has a much broader scope for neutron protein crystallography and which is capable of providing good results for systems having larger unit cells and smaller crystal volumes. The isotopic replacement of hydrogen by deuterium atoms allows their identification in neutron scattering density maps, with a visibility comparable to that of other elements in biological macromolecules, as well as helping to reduce the high experimental background due to the large incoherent scattering cross-section of hydrogen. [6] Perdeuterated protein was produced in the Deuteration Laboratory of ILLs Life Science Group; its use allows both the elimination of hydrogen incoherent scattering and also the enhancement of the visibility of deuterium by comparison with its lighter isotope hydrogen. The study was carried out to a resolution of 1.27 in the case of the oxidized form (PDB: 4AR3) and to 1.38 resolution for the reduced form (PDB: 4AR4). This is the first high-resolution neutron study of Pf rubredoxin in both forms. It reveals information beyond that attainable in previous lower-resolution studies of hydrogenated [7] and deuterated systems. [8] The combination of monochromatic neutron crystallography and protein perdeuteration has played a key role in obtaining this high-quality structural information.Our neutron ...

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DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly.

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The endonuclease activity within the influenza virus cap-snatching process is a proven therapeutic target. The anti-influenza drug baloxavir is highly effective, but is associated with resistance mutations that threaten its clinical efficacy. The endonuclease resides within the N-terminal domain of the PA subunit (PAN) of the influenza RNA dependent RNA polymerase, and we report here complexes of PAN with RNA and DNA oligonucleotides to understand its specificity and the structural basis of baloxavir resistance mutations. The RNA and DNA oligonucleotides bind within the substrate binding groove of PAN in a similar fashion, explaining the ability of the enzyme to cleave both substrates. The individual nucleotides occupy adjacent conserved pockets that flank the two-metal active site. However, the 2′ OH of the RNA ribose moieties engage in additional interactions that appear to optimize the binding and cleavage efficiency for the natural substrate. The major baloxavir resistance mutation at position 38 is at the core of the substrate binding site, but structural studies and modeling suggest that it maintains the necessary virus fitness via compensating interactions with RNA. These studies will facilitate the development of new influenza therapeutics that spatially match the substrate and are less likely to elicit resistance mutations.