Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis

The nature of the interaction of the transition-state analogue inhibitor L-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a K(i) of 6.6 microM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 A resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an eta-1,2-mu-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.     

School screening for scoliosis: cohort study of clinical course.

A visual examination of 1764 Oxford schoolchildren for scoliosis was followed by low-dose radiography of the spine in those who showed evidence of asymmetric body topography; radiography was repeated a year later to assess progression. Forty-four children had curves of 10 degrees or more. Two had a congenital abnormality and the remaining 42 were classified according to the type of curve: sacral tilt (compensatory), spinal (idiopathic), or combined (sacral tilt and spinal). Progression occurred in 6 (14%) children, none of whom had only a sacral tilt. These results suggest that only by measuring sacral tilt can benign compensatory curves be differentiated from true idiopathic scoliosis.     

tigaR: integrative significance analysis of temporal differential gene expression induced by genomic abnormalities

Background

To determine which changes in the host cell genome are crucial for cervical carcinogenesis, a longitudinal in vitro model system of HPV-transformed keratinocytes was profiled in a genome-wide manner. Four cell lines affected with either HPV16 or HPV18 were assayed at 8 sequential time points for gene expression (mRNA) and gene copy number (DNA) using high-resolution microarrays. Available methods for temporal differential expression analysis are not designed for integrative genomic studies.

Results

Here, we present a method that allows for the identification of differential gene expression associated with DNA copy number changes over time. The temporal variation in gene expression is described by a generalized linear mixed model employing low-rank thin-plate splines. Model parameters are estimated with an empirical Bayes procedure, which exploits integrated nested Laplace approximation for fast computation. Iteratively, posteriors of hyperparameters and model parameters are estimated. The empirical Bayes procedure shrinks multiple dispersion-related parameters. Shrinkage leads to more stable estimates of the model parameters, better control of false positives and improvement of reproducibility. In addition, to make estimates of the DNA copy number more stable, model parameters are also estimated in a multivariate way using triplets of features, imposing a spatial prior for the copy number effect.

Conclusion

With the proposed method for analysis of time-course multilevel molecular data, more profound insight may be gained through the identification of temporal differential expression induced by DNA copy number abnormalities. In particular, in the analysis of an integrative oncogenomics study with a time-course set-up our method finds genes previously reported to be involved in cervical carcinogenesis. Furthermore, the proposed method yields improvements in sensitivity, specificity and reproducibility compared to existing methods. Finally, the proposed method is able to handle count (RNAseq) data from time course experiments as is shown on a real data set.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-327) contains supplementary material, which is available to authorized users.     

Spectroscopic and X-ray crystallographic characterization of bestatin bound to the aminopeptidase from Aeromonas (Vibrio) proteolytica

Binding of the competitive, slow-binding inhibitor bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from Aeromonas proteolytica (AAP) was examined by both spectroscopic and crystallographic methods. Electronic absorption spectra of the catalytically competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the presence of bestatin revealed that both of the divalent metal ions in AAP are involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum of the [CoCo(AAP)]-bestatin complex exhibited no observable perpendicular- or parallel-mode signal. These data indicate that the two Co(II) ions in AAP are antiferromagnetically coupled yielding an S = 0 ground state and suggest that a single oxygen atom bridges between the two divalent metal ions. The EPR data obtained for [CoZn(AAP)] and [ZnCo(AAP)] confirm that bestatin interacts with both metal ions. The X-ray crystal structure of the [ZnZn(AAP)]-bestatin complex was solved to 2.0 A resolution. Both side chains of bestatin occupy a well-defined hydrophobic pocket that is adjacent to the dinuclear Zn(II) active site. The amino acid residues ligated to the dizinc(II) cluster in AAP are identical to those in the native structure with only minor perturbations in bond length. The alkoxide oxygen of bestatin bridges between the two Zn(II) ions in the active site, displacing the bridging water molecule observed in the native [ZnZn(AAP)] structure. The M-M distances observed in the AAP-bestatin complex and native AAP are identical (3.5 A) with alkoxide oxygen atom distances of 2.1 and 1.9 A from Zn1 and Zn2, respectively. Interestingly, the backbone carbonyl oxygen atom of bestatin is coordinated to Znl at a distance of 2.3 A. In addition, the NH(2) group of bestatin, which mimics the N-terminal amine group of an incoming peptide, binds to Zn2 with a bond distance of 2.3 A. A combination of the spectroscopic and X-ray crystallographic data presented herein with the previously reported mechanistic data for AAP has provided additional insight into the substrate-binding step of peptide hydrolysis as well as insight into important small molecule features for inhibitor design.     

Structure of MurF from Streptococcus pneumoniae co-crystallized with a small molecule inhibitor exhibits interdomain closure

In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.