Bound Heat No Escape 2

Bound Heat No Escape 2

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Brody stood behind the students, rested his hands on his hips and shifted his gaze from the male climber to the woman. Her chalked fingers clung to slivers of manufactured rock while her feet perched on similar pieces. Tight black pants and a white, fitted spandex top molded a trim athletic body. Long, red hair bound into a ponytail swept across her muscled back as she scrambled haphazardly from rock to rock. Jo? He looked closer.

"Sure." He shut off the engine and followed her up the sidewalk, cracked in spots by last summer's heat. (Continues...) Excerpted from No Escape by MARY BURTON. Copyright © 2013 Mary Burton. Excerpted by permission of KENSINGTON PUBLISHING CORP.. All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

The structure of the HIV-1 RT shown along the template/primer binding cleft in the (a) closed apo (based on the 1DLO crystal structure) and (b) open NNRTI bound conformation (based on the EFV bound 1IKW structure, the van der Waals surface of the drug is shown). In this view the p66 subunit is most prominent, in (c) viewed from above the template/primer binding cleft both the p66 and p51 monomers are clearly visible. The subunits are named after the structures supposed likeness to a right hand. It is in fact, the folding of the p66 seen in (a) and (b) which results in the likeness. However the subdomains retain their name in p51 as seen in (b). The subdomains are known as the (F)ingers (blue), (P)alm (red), (T)humb (green), (C)onnection (yellow) and (R)NaseH (orange). The first 4 subdomains form the polymerase domain and are common to both subunits. In all figures the residues of the polymerase and RNaseH active sites are shown as magenta beads (the former is located in the palm). The residues of the RNaseH primer grip are indicated by black beads.

HIV-1 RT is a heterodimeric protein consisting of 440 and 560 amino acid subunits (known as p51 and p66 respectively due to their molecular weights). Both subunits are derived from the same gag-pol polyprotein with the p51 subunit lacking the C-terminal domain (containing the RNaseH active site), which is cleaved from it by the HIV-1 protease. The PDB [3] contains many different crystal structures of HIV-1 RT in a range of states including those bound to both NRTIs [4,5] and NNRTIs [6,7,8], natural DNA and RNA substrates [9,10,11,12] in addition to the apoenzyme [13]. The supposed resemblance of the p66 to a right hand has led to the shared subdomains being known as the fingers, palm, thumb and connection [14]. The reported crystal structures of the unliganded enzyme show the tips of the p66 fingers and thumb domains in close proximity, with a separation of less than 15 Å (one structure does not exhibit this feature but was created by soaking out an NNRTI), in what is known as the closed conformation. Despite deriving from the same sequence the subunits have very different forms once folded. All template/primer bound systems show the enzyme in a more open configuration with the template/primer substrate fitting between the thumb and fingers domains [15]. A similar conformation, but with greater separation between the p66 fingers and thumb, is seen in all NNRTI bound structures. Figure 1 shows both the closed apo and open NNRTI bound conformations of the enzyme. Both the polymerase and RNase H catalytic sites are located within the p66 subunit. The polymerase active site (formed by Asp110, Asp185 and Asp186) is found in the palm subdomain, the RNaseH active site being located at the opposite end of the enzyme with a distance of approximately 30 Å separating them along the central template/primer binding cleft. When bound to a RNA/DNA hybrid template/primer approximately 19 base pairs are contained between the two catalytic sites [12]. The template/primer is positioned at the two active sites by contacts with regions known as the polymerase primer grip (p66 residues 227 to 235) and the RNaseH primer grip (p66 residues 359, 360, 361, 473, 475, 476, 501 and 505, and p51 residues 395 and 396) [12].

Comparison of the first 15 ANM modes generated from the apo HIV-1 RT structure and those of the NVP and EFV bound enzyme using the 1DLO, 1RTH and 1FK9 crystal structures respectively. The matrices show the pairwise correlation of modes, the darker the square the higher the magnitude of the correlation. (a) The apo to NVP bound and (b) apo to EFV bound comparisons show similar patterns with high correlations between the first and third modes and lower correlation in most other modes. (c) shows that the modes for the EFV and NVP bound structures are much more similar. The correlation of mode 2 for both structures with other modes indicates that this mode is not identical between the two structures.

The fluctuations of the NNRTI bound HIV-1 RT described by the second ANM mode for (a) the EFV bound 1FK9 and (b) NVP bound 1RTH structures. The fluctuations of each residue are shown as magenta arrows. In both cases two views are shown, one above the other; along the DNA binding cleft and looking down into it. In both cases the motions are dominated by anti-correlated movements of the p66 thumb and fingers (as highlighted by the black arrows).

Comparison of the first 15 ANM modes generated from different HIV-1 RT structures bound to NVP and EFV, using the 1RTH and 1VRT NVP bound structures and 1FK9 and 1IKW EFV bound structures. The matrices show the magnitude of the pairwise correlation of the modes, the darker the square the higher the correlation. (a) and (b) show comparisons of the modes generated from different crystal structures but with RT bound to the same inhibitor, NVP and EFV respectively. In (c) the 1IKW (EFV) and 1VRT (NVP) bound structures are compared, more of the low modes show high levels of similarity than in the plot for 1FK9 and 1RTH shown in Figure 2c.

Comparison of the squared fluctuations predicted by ANM mode 2 of the NVP bound 1VRT (red) and EFV bound 1IKW structures (black). The main difference between the two structures is in the extent of the predicted mobility of the p66 thumb (residues 244 to 322); in both cases this is much than that observed for 1RTH and 1FK9 (see Figure 5).

If there is no link between the specific inhibitor bound and the differences seen in the ANM modes of NNRTI bound HIV-1 RT, the obvious question is which features of the structures are causing the observed variation. In order to assess these differences observed in the ANMs we used PCA to analyse the differences in the available EFV and NVP bound structures. Analysis of all available crystal structures revealed one prominent principal component (PC), but this only identified differences between the 3HVT structure which was crystalised in the C2 space group and the other crystal structures all in the P 212121 space group. This difference is known to produce alterations in RT conformation [48] and the fact this structure is also anomalous in not having a water molecule bound beside NVP led us to exclude it from consideration here. The first PC generated for the reduced dataset accounts for 48% of the variance observed and the second a further 20% (the variance accounted for by the first 20 PCs is shown in Supporting Information Supplementary Figure S4). The projection of each crystal structure analysed onto these two components is shown in Figure 8. Two separate clusters of structures are apparent in this data, where we have labelled the two groups A and B. All structures in group A (including 1FK9,1IKW and 1VRT) show p51 flexibility in their second ANM modes, whereas those in group B do not. A list of structures in each group is provided in Table 1. Both groups contain both EFV and NVP structures and both wild type and resistant sequences (although NVP bound structures with the key NNRTIBP mutations Y181C and Y188C are all found in group B). The two structures separated from the main group A along PC2 cluster (Figure 8) are 1IKV and 1IKW. The variation along PC2 is dominated by motions in highly flexible loops in the p66 fingers (see Supporting Information Supplementary Figure S5). As these two structures were produced in the same study [7] it is likely that these minor changes are a function of the particular crystallization protocol employed.

Projection of individual crystal structures on to the first two principal components generated from PCA of EFV and NVP bound HIV-1 RT structures. The structures are clearly clustered into two groups along PC1, labelled A and B, and highlighted in red and blue respectively.

The EFV and NVP bound HIV-1 RT crystal structures analysed by PCA are divided into two groups according to PC1, groups A and B. Group A corresponds to the structures in which p51 flexibility is linked to that of the p66 thumb by ANMs, no such link is observed in ANMs based on group B structures. The drug bound and major resistance associated mutations present in the structure are also listed.

The variation of the NNRTI bound HIV-1 RT described by PC1. The variation of each residue, relative to the average structure displayed in tube representation, is shown in (a) as magenta arrows. Detail of the changes in their p66 thumb is shown in (b), the Cα atoms of residue 270 (black) and 348 (cyan) are shown as spheres.

Projection of snapshots from the ensemble MD trajectories of the EFV and NVP bound HIV-1 RT onto the first two principal components generated from PCA of EFV and NVP bound HIV-1 RT crystal structures. Both simulations show wide variations in both PCs; there is no clustering close to either of the groups of crystal structure projections (these are shown by the dashed rectangles and labelled A and B). Crystal structure projections are shown as black dots, except the 1IKW (green) and 1VRT (magenta) structures which were used as initial structures for the EFV and NVP MD simulations, respectively. 2b1af7f3a8