Crystal structure of the arginine repressor protein in complex with the DNA operator from Mycobacterium tuberculosis

The arginine repressor (ArgR) from Mycobacterium tuberculosis (Mtb) is a gene product encoded by the open reading frame Rv1657. It regulates the l-arginine concentration in cells by interacting with ARG boxes in the promoter regions of the arginine biosynthesis and catabolism operons. Here we present a 2.5-Å structure of MtbArgR in complex with a 16-bp DNA operator in the absence of arginine. A biological trimer of the protein-DNA complex is formed via the crystallographic 3-fold symmetry axis. The N-terminal domain of MtbArgR has a winged helix-turn-helix motif that binds to the major groove of the DNA. This structure shows that, in the absence of arginine, the ArgR trimer can bind three ARG box half-sites. It also reveals the structure of the whole MtbArgR molecule itself containing both N-terminal and C-terminal domains.     

Tet repressor induction by tetracycline: a molecular dynamics, continuum electrostatics, and crystallographic study

The Tet repressor (TetR) mediates the most important mechanism of bacterial resistance against tetracycline (Tc) antibiotics. In the absence of Tc, TetR is tightly bound to its operator DNA; upon binding of Tc with an associated Mg²⁺ ion, it dissociates from the DNA, allowing expression of the repressed genes. Its tight control by Tc makes TetR broadly useful in genetic engineering. The Tc binding site is over 20 Å from the DNA, so the binding signal must propagate a long distance. We use molecular dynamics simulations and continuum electrostatic calculations to test two models of the allosteric mechanism. We simulate the TetR:DNA complex, the Tc-bound, “induced” TetR, and the transition pathway between them. The simulations support the model inferred previously from the crystal structures and reveal new details. When [Tc:Mg]⁺ binds, the Mg²⁺ ion makes direct and water-mediated interactions with helix 8 of one TetR monomer and helix 6 of the other monomer, and helix 6 is pulled in towards the central core of the structure. Hydrophobic interactions with helix 6 then pull helix 4 in a pendulum motion, with a maximal displacement at its N-terminus: the DNA interface. The crystal structure of an additional TetR reported here corroborates this motion. The N-terminal residue of helix 4, Lys48, is highly conserved in DNA-binding regulatory proteins of the TetR class and makes the largest contribution of any amino acid to the TetR:DNA binding free energy. Thus, the conformational changes lead to a drastic reduction in the TetR:DNA binding affinity, allowing TetR to detach itself from the DNA. Tc plays the role of a specific Mg²⁺ carrier, whereas the Mg²⁺ ion itself makes key interactions that trigger the allosteric transition in the TetR:Tc complex.     

A conserved dimer and global conformational changes in the structure of apo-PknE Ser/Thr protein kinase from Mycobacterium tuberculosis

The "eukaryotic-like" receptor Ser/Thr protein kinases (STPKs) are candidates for the sensors that mediate environmental adaptations of Mycobacterium tuberculosis (Mtb). To define the mechanisms of regulation and substrate recognition, we determined the crystal structure of the ligand-free, activated kinase domain (KD) of the Mtb STPK, PknE. Remarkably, the PknE KD formed a dimer similar to that first observed in the structure of the ATPgammaS complex of the Mtb paralog, PknB. This structural similarity, which occurs despite little sequence conservation between the PknB and PknE dimer interfaces, supports the idea that dimerization regulates the Mtb receptor STPKs. Insertion of the DFG motif into the ATP-binding site and other conformational differences compared the ATPgammaS:PknB complex suggest that apo-PknE is not pre-organized to bind nucleotides. This structure may represent an inactive conformation stabilized by dimerization or, alternatively, an active conformation that reveals shifts that mediate nucleotide exchange and order substrate binding.     

An NMR-based molecular dynamics simulation of the interaction of the lac repressor headpiece and its operator in aqueous solution

The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complex satisfies essentially all experimental distance information derived from two-dimensional nuclear magnetic resonance (2-D-NMR) studies. The interaction between lac repressor headpiece and its operator is based on many direct- and water-mediated hydrogen bonds and nonpolar contacts which allow the formation of a tight complex. No stable hydrogen bonds between side chains and bases are found, while specific contacts occur between both nonpolar groups and, to a lesser extent, through water-mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, and chemically induced dynamic nuclear polarization (CIDNP) data.     

Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.     

Determination of the secondary structure in solution of the Escherichia coli DnaA DNA-binding domain

DnaA protein binds specifically to a group of binding sites collectively called as DnaA boxes within the bacterial replication origin to induce local unwinding of duplex DNA. The DNA-binding domain of DnaA, domain IV, comprises the C-terminal 94 amino acid residues of the protein. We overproduced and purified a protein containing only this domain plus a methionine residue. This protein was stable as a monomer and maintained DnaA box-specific binding activity. We then analyzed its solution structure by CD spectrum and heteronuclear multi-dimensional NMR experiments. We established extensive assignments of the 1H, 13C, and 15N nuclei, and revealed by obtaining combined analyses of chemical shift index and NOE connectivities that DnaA domain IV contains six alpha-helices and no beta-sheets, consistent with results of CD analysis. Mutations known to reduce DnaA box-binding activity were specifically located in or near two of the alpha-helices. These findings indicate that the DNA-binding fold of DnaA domain IV is unique among origin-binding proteins.     

The solution structure of the oxidative stress-related protein YggX from Escherichia coli

YggX is a highly conserved protein found only in eubacteria and is proposed to be involved in the bacterial response to oxidative stress. Here we report the solution structure of YggX from Escherichia coli determined by nuclear magnetic resonance spectroscopy. The structure of YggX displays a fold consisting of two N-terminal antiparallel beta-sheets and three alpha-helices, which shares significant structural similarity to the crystal structure of a hypothetical protein PA5148 from Pseudomonas aeruginosa. Previous studies propose YggX as an iron binding protein that is involved in cellular iron trafficking. Our data indicate that the protein alone does not bind iron in vitro, suggesting other cofactors or different conditions may be necessary for metal binding.     

Holarctic genetic structure and range dynamics in the woolly mammoth

Ancient DNA analyses have provided enhanced resolution of population histories in many Pleistocene taxa. However, most studies are spatially restricted, making inference of species-level biogeographic histories difficult. Here, we analyse mitochondrial DNA (mtDNA) variation in the woolly mammoth from across its Holarctic range to reconstruct its history over the last 200 thousand years (kyr). We identify a previously undocumented major mtDNA lineage in Europe, which was replaced by another major mtDNA lineage 32–34 kyr before present (BP). Coalescent simulations provide support for demographic expansions at approximately 121 kyr BP, suggesting that the previous interglacial was an important driver for demography and intraspecific genetic divergence. Furthermore, our results suggest an expansion into Eurasia from America around 66 kyr BP, coinciding with the first exposure of the Bering Land Bridge during the Late Pleistocene. Bayesian inference indicates Late Pleistocene demographic stability until 20–15 kyr BP, when a severe population size decline occurred.     

Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae

The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria. VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera. We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt). VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data. The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions. Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain. Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.     

How the timberline formed: altitudinal changes in stand structure and dynamics around the timberline in central Japan

Background and Aims

Altitudinal timberlines are thought to move upward by global warming, a crucial topic in ecology. Tall tree species (the conifer Abies mariesii and the deciduous broad-leaved Betula ermanii) dominate the sub-alpine zone between 1600 and 2500 m a.s.l., the timberline, on Mount Norikura in central Japan. Dwarf pine Pinus pumila dominates above the timberline to near the summit (3026 m a.s.l.). This study evaluated how the timberline formed on Mount Norikura by examining altitudinal changes in stand structure and dynamics around the timberline.

Methods

One hundred and twenty-five plots of 10 m × 10 m were established around the timberline (2350–2600 m a.s.l.). Trunk diameter growth rate during 6 years was examined for A. mariesii, B. ermanii and P. pumila. Mortality during this period and mechanical damage scars on the trunks and branches due to strong wind and snow were examined for A. mariesii only.

Key Results

The density, maximum trunk height and diameter of A. mariesii in plots decreased with altitude. The maximum trunk height of B. ermanii decreased with altitude, but density and maximum trunk diameter did not decrease. In contrast, the density of P. pumila abruptly increased from around the timberline. A strong negative correlation was found between the densities of P. pumila and tall tree species, indicating their interspecific competition. Trunk diameter growth rates of A. mariesii and B. ermanii did not decrease with altitude, suggesting that these two tall tree species can grow at the timberline. The ratio of trees with mechanical damage scars increased with altitude for A. mariesii, a tendency more conspicuous for larger trees. The mortality of larger A. mariesii was also greater at higher altitude. Tall tree species may not increase their trunk height and survive around the timberline because of mechanical damage.

Conclusions

This study suggests that the altitudinal location of the timberline is mainly affected by mechanical damage due to strong wind and snow rather than by growth limitation due to low temperature. Therefore, the timberline would not move upward even under global warming if these growth and mortality characteristics do not change for a long time.     

Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor

Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported; those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.     

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.