Cooperative fluctuations of unliganded and substrate-bound HIV-1 protease: a structure-based analysis on a variety of conformations from crystallography and molecular dynamics simulations

The dynamics of HIV-1 protease, both in unliganded and substrate-bound forms have been analyzed by using an analytical method, Gaussian network model (GNM). The method is applied to different conformations accessible to the protein backbone in the native state, observed in crystal structures and snapshots from fully atomistic molecular dynamics (MD) simulation trajectories. The modes of motion obtained from GNM on different conformations of HIV-1 protease are conserved throughout the MD simulations. The flaps and 40's loop of the unliganded HIV-1 protease structure are identified as the most mobile regions. However, in the liganded structure these flaps lose mobility, and terminal regions of the monomers become more flexible. Analysis of the fast modes shows that residues important for stability are in the same regions of all the structures examined. Among these, Gly86 appears to be a key residue for stability. The contribution of residues in the active site region and flaps to the stability is more pronounced in the substrate-bound form than in the unliganded form. The convergence of modes in GNM to similar regions of HIV-1 protease, regardless of the conformation of the protein, supports the robustness of GNM as a potentially useful and predictive tool.     

Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles

In NMR studies of large molecular structures, the number of conformational constraints based on NOE measurements is typically limited due to the need for partial deuteration. As a consequence, when using selective protonation of peripheral methyl groups on a perdeuterated background, stereospecific assignments of the diastereotopic methyl groups of Val and Leu can have a particularly large impact on the quality of the NMR structure determination. For example, 3D ¹⁵N- and ¹³C-resolved [¹H,¹H]-NOESY spectra of the E. Coli membrane protein OmpX in mixed micelles with DHPC, which have an overall molecular weight of about 60 kDa, showed that about 50% of all obtainable NOEs involve the diastereotopic methyl groups of Val and Leu. In this paper, we used biosynthetically-directed fractional ¹³C labeling of OmpX and [¹³C,¹H]-HSQC spectroscopy to obtain stereospecific methyl assignments of Val and Leu in OmpX/DHPC. For practical purposes it is of interest that this data could be obtained without use of a deuterated background, and that combinations of NMR experiments have been found for obtaining the desired information either at a ¹H frequency of 500 MHz, or with significantly reduced measuring time on a high-frequency instrument.     

Crystal and molecular structure of methyl 4-O-methyl-beta-D-ribo-hex-3-ulopyranoside

Methyl 4-O-methyl-beta-D-ribo-hex-3-ulopyranoside (2), a model compound for partially oxidized anhydroglucose units in cellulose, was crystallized from CHCl(3)/n-hexane by vapor diffusion to give colorless plates. Crystal structure determination revealed the monoclinic space group P2(1) with Z = 2C(8)H(14)O(6) and unit cell parameters of a = 8.404(2), b = 4.5716(10), c = 13.916(3)A, and beta = 107.467(4) degrees. The structure was solved by direct methods and refined to R = 0.0476 for 1655 reflections and 135 parameters. The hexulopyranoside occurs in a distorted chair conformation. Both hydroxyls are involved in hydrogen bonding and form zigzag bond chains along the b-axis. One of the two hydrogen bonds is bifurcated. The solid-state (13)C NMR spectrum of exhibits eight carbon resonances, with well-separated signals for the two methoxyls (1-OMe: 55.72 ppm, 4-OMe: 61.25 ppm) and a keto resonance with relatively large downfield shift (206.90 ppm). Differences in the C-4 and the methoxyls' chemical shifts in the solid and liquid states were found.     

Phylogenetic analysis of the vertebrate galectin family

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.     

How meaningful are Bayesian support values?

In this study, we used an empirical example based on 100 mitochondrial genomes from higher teleost fishes to compare the accuracy of parsimony-based jackknife values with Bayesian support values. Phylogenetic analyses of 366 partitions, using differential taxon and character sampling from the entire data matrix of 100 taxa and 7,990 characters, were performed for both phylogenetic methods. The tree topology and branch-support values from each partition were compared with the tree inferred from all taxa and characters. Using this approach, we quantified the accuracy of the branch-support values assigned by the jackknife and Bayesian methods, with respect to each of 15 basal clades. In comparing the jackknife and Bayesian methods, we found that (1) both measures of support differ significantly from an ideal support index; (2) the jackknife underestimated support values; (3) the Bayesian method consistently overestimated support; (4) the magnitude by which Bayesian values overestimate support exceeds the magnitude by which the jackknife underestimates support; and (5) both methods performed poorly when taxon sampling was increased and character sampling was not increases. These results indicate that (1) the higher Bayesian support values are inappropriate (in magnitude), and (2) Bayesian support values should not be interpreted as probabilities that clades are correctly resolved. We advocate the continued use of the relatively conservative bootstrap and jackknife approaches to estimating branch support rather than the more extreme overestimates provided by the Markov Chain Monte Carlo-based Bayesian methods.     

Comparison of C-erbB2 (HER-2/Neu) immunohistochemistry results on invasive breast carcinomas. Experience of 2 Pathology departments in Turkey and Greece

OBJECTIVE: To compare c-erbB2 (HER-2/Neu) immunohistochemical staining results obtained in 2 pathology departments and evaluate the reproducibility of staining and assessments. STUDY DESIGN: Ninety primary invasive ductal carcinoma cases constituted the material of this study. For concordant assessment, serial sections from the same tissue blocks were prepared in both Turkey and Greece and were sent to each other. Evaluation of c-erbB2 (HER-2/Neu) staining was done independently in 2 departments by experienced pathologists. Stained slides were evaluated semiquantitatively in Turkey and both semiquantitatively and automatically in Greece. RESULTS: There was complete agreement on staining density in 58 (64.4%) cases in both centers. In 15 cases there were major discordances in staining degrees, and in 17 cases there was a major discordance. Hypothetical treatment decisions based on these results showed that 82% of patients would have been handled the same way. CONCLUSION: Comparison of immunostaining patterns performed at 2 centers provided valuable data that may be used in the development of quality assurance policies. The present study showed the usefulness of multicenter comparative studies in initiating the development of guidelines.     

Letter to the Editor: NMR Structure of a Variant 434 Repressor DNA-binding Domain Devoid of Hydroxyl Groups

Abbreviations: 434(1–63) – N-terminal 63-residue DNA-binding domain of the phage 434 repressor; dh434(0–63) – variant 434 repressor DNA-binding domain devoid of hydroxyl groups; P22c2(1–76) – N-terminal 76-residue fragment of the phage P22 c2 repressor; SDS-PAGE – SDS polyacrylamide gel electrophoresis; COSY – correlation spectroscopy; TOCSY – total correlation spectroscopy; NOE – nuclear Overhauser effect; RMSD – root-mean-square deviation.