Conformational switch of RegX3 active Pocket in MD simulation.

<p>(A) Structural alignment of simulated RegX3s (colored in orange) and original active RegX3 (colored in green). The alignment was based on core folding of ED. α4β5α5 face elements are labeled. Rotation degree of α5 is indicated with arrow in the lower half of the structure. Perpendicular and parallel switch for β4α4 are marked in the upper half of the structure. (B) High mobility of RegX3 residues based root-mean-square fluctuation (rmsf). (C) Active pocket stabilized by key residues together with a lanthanum ion in the active conformation of RegX3 in comparison with simulated RegX3s. The alignment and color scheme are the same as panel A. Coupled residues Thr⁷⁹ and Tyr⁹⁸ together with other important residues around active site are labeled with sticks. The green sphere represents Lanthanum ion.</p

Conformational Dynamics of Response Regulator RegX3 from <i>Mycobacterium tuberculosis</i>

<div><p>Two-component signal transduction systems (TCS) are vital for adaptive responses to various environmental stresses in bacteria, fungi and even plants. A TCS typically comprises of a sensor histidine kinase (SK) with its cognate response regulator (RR), which often has two domains—N terminal receiver domain (RD) and C terminal effector domain (ED). The histidine kinase phosphorylates the RD to activate the ED by promoting dimerization. However, despite significant progress on structural studies, how RR transmits activation signal from RD to ED remains elusive. Here we analyzed active to inactive transition process of OmpR/PhoB family using an active conformation of RegX3 from <i>Mycobacterium tuberculosis</i> as a model system by computational approaches. An inactive state of RegX3 generated from 150 ns molecular dynamic simulation has rotameric conformations of Thr⁷⁹ and Tyr⁹⁸ that are generally conserved in inactive RRs. Arg⁸¹ in loop β4α4 acts synergistically with loop β1α1 to change its interaction partners during active to inactive transition, potentially leading to the N-terminal movement of RegX3 helix α1. Global conformational dynamics of RegX3 is mainly dependent on α4β5 region, in particular seven ‘hot-spot’ residues (Tyr⁹⁸ to Ser¹⁰⁴), adjacent to which several coevolved residues at dimeric interface, including Ile⁷⁶-Asp⁹⁶, Asp⁹⁷-Arg¹¹¹ and Glu²⁴-Arg¹¹³ pairs, are critical for signal transduction. Taken together, our computational analyses suggest a molecular linkage between Asp phosphorylation, proximal loops and α4β5α5 dimeric interface during RR active to inactive state transition, which is not often evidently defined from static crystal structures.</p></div

In silico mutagenesis analysis of RegX3.

<p>(A) Simulated structures of active RegX3 with introduced mutations are analyzed by PCA. RegX3s and RegX3-5ns used as a control are those simulated structures after 150 ns and 5 ns, respectively. mRegX3 indicates RegX3 mutant simulated for 5 ns with numbers in parenthesis for alanine mutation of specific sites. DrrB and DrrD are representative inactive and open RR structures with exposed DNA recognition helices. MtrA and PrrA are representative inactive and compact structures with buried DNA recognition helices. Structures are grouped by color dots. (B) The frequency distribution of individual residues contributing to all RegX3 structures on PC spaces. Y-axis indicates the observed frequency and X-axis represents the residue numbers of RegX3. Grey and black color regions on the X-axis indicate β sheets and α helices whereas the white spaces in between indicate loop regions.</p

Interdomain interface between receiver and effector domains.

<p>Interdomain interactions between RD and ED domains of original active state (A) and simulated inactive state RegX3 (B) are shown in cartoon. The top half of each panel is from the ED in orange; the lower half is α5 and its adjacent loop in grey from the RD. Sticks represent all interaction residues with hydrogen bonds in yellow dotted lines.</p

Coevolved residues of dimer interface of OmpR/PhoB family calculated by GREMLIN: Listed are the interacting residues with their respective numberings and topology.

<p>Italicized pairs indicate coevolved inter-monomeric contacts of RegX3 but intra-monomeric contacts in other OmpR/PhoB family members. Bold representation highlights those dimeric interactions conserved in all OmpR/PhoB family members. Contact pair with asterisk symbol satisfies all available active RR structures in this family but not RegX3.</p

Coevolution analysis of RegX3.

<p>Contact map of coevolved residues in OmpR/PhoB family. Axes are residue numbers. Grey spots represent contacts within the same monomer, while black spots are predicted inter-monomeric contacts. The blue spots highlight those coevolved pairs in dimeric interface listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133389#pone.0133389.t001" target="_blank">Table 1</a>.</p

Coevolved residues in dimeric interface of OmpR/PhoB family.

<p>(A) Sequence conservation of the α4β5α5 dimeric interface of RegX3, PhoB, TorR and YycF in OmpR/PhoB family. Residues are numbered according to RegX3 and colored based on chemistry: acidic in red, basic in blue, hydrophobic in black, polar in green and neutral in purple. Coevolved pairs are under-dotted with the same colors. (B-E) Active state structures of RegX3 (2OQR), TorR (1ZGZ), PhoB (1ZES) and YycF (1NXP) in OmpR/PhoB family with coevolved residues in dimeric interface highlighted in sticks. It is noteworthy that RegX3 forms a complete dimeric structure with swapped domains that are colored in light and dark greys.</p

Normal model analysis for conformational dependent regions of RegX3.

<p>(A) Overlap score of all non-zero low frequency modes between active and inactive state structures. (B) Overlap score of non-zero ENM modes based on the contribution of pocket residues of α4β5α5 face to the overall changes of full-length RegX3. (C) Contribution fraction (Y-axis) of residues to low frequency NMA modes in pocket residue analysis. (D) Key structural region with the nature of individual residues contributes to conformational changes during active to inactive transition of RegX3. ED is colored in grey and RD in blue. Helix α4 is colored in green and hot spot residues are in sphere models. The direction of an arrow represents the hinge motion of blue (ED) towards black (RD).</p

Dynamics of β1α1, β3α3 and β4α4 loops.

<p>(A-C) rmsd deviation plots of β1α1, β3α3 and β4α4 loops of RegX3 during 150 ns simulation. X-axis is time step and Y-axis indicates rmsd deviations. Interaction networks of these loops are explicated in active state (D) and inactive state (E). Residues are shown in ball and sticks. Brown and green spheres indicate La³⁺ ion and water molecule, respectively. All interactions are highlighted in dashed lines.</p

DataSheet2_The Changes in Drainage Systems of Weihe Basin and Sanmenxia Basin Since Late Pliocene Give New Insights Into the Evolution of the Yellow River.xlsx

The formation of the Yellow River involved the draining of a series of ancestral local lakes along their course, substantially changing the regional, geomorphic, and paleoenvironmental evolution. However, the evolution of the Weihe-Sanmenxia Basin section of the Yellow River remains indistinct as previous studies regard the Weihe and Sanmenxia Basin as one integral basin of the Late Cenozoic. Here, we present the detrital zircon age spectra from the Pliocene-Pleistocene Sanmen Formation to clarify the drainage system evolution of the two basins since the Late Pliocene. The results reveal that these two basins belonged to different drainage systems in the Late Pliocene because no sediments from the marginal mountains of the Weihe Basin accumulated in the Sanmenxia Basin. At 2.8/2.6 Ma, the currents presented at the edge of the basins and transported the sediment of east Hua Mountain into the Sanmenxia Basin, where it was deposited. This integration likely leads to a mismatch between the deposition and regional paleoclimate in previous studies. At ∼1.0 Ma, the Sanmenxia Gorge was traversed and the Yellow River finally formed, depositing Jinshaan Gorge sediment into the Sanmenxia Basin and lower reaches of the Yellow River.</p