LabCaS: Labeling calpain substrate cleavage sites from amino acid sequence using conditional random fields

The calpain family of Ca²⁺‐dependent cysteine proteases plays a vital role in many important biological processes which is closely related with a variety of pathological states. Activated calpains selectively cleave relevant substrates at specific cleavage sites, yielding multiple fragments that can have different functions from the intact substrate protein. Until now, our knowledge about the calpain functions and their substrate cleavage mechanisms are limited because the experimental determination and validation on calpain binding are usually laborious and expensive. In this work, we aim to develop a new computational approach (LabCaS) for accurate prediction of the calpain substrate cleavage sites from amino acid sequences. To overcome the imbalance of negative and positive samples in the machine‐learning training which have been suffered by most of the former approaches when splitting sequences into short peptides, we designed a conditional random field algorithm that can label the potential cleavage sites directly from the entire sequences. By integrating the multiple amino acid features and those derived from sequences, LabCaS achieves an accurate recognition of the cleave sites for most calpain proteins. In a jackknife test on a set of 129 benchmark proteins, LabCaS generates an AUC score 0.862. The LabCaS program is freely available at: http://www.csbio.sjtu.edu.cn/bioinf/LabCaS . Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

Molecular docking simulation analysis of alcohol acyltransferases from two related fruit species explains their different substrate selectivities

Tropical papaya (Carica papaya) and mountain papaya (Vasconcellea pubescens) fruits are characterised for their strong and particular aroma. The aroma of both fruits is different and dominated by esters, which are synthesised by alcohol acyltransferases (AATs). The ability to produce esters is contrasting, V. pubescens (VpAAT1) being a very active enzyme towards the production of benzyl acetate, whereas C. papaya (CpAAT1) is more active towards the production of ethyl butanoate and methyl butanoate, but not benzyl acetate. In order to understand the mechanism of action at the molecular level, the structural model of CpAAT1 protein was built by comparative modelling. Conformational interaction between the protein and several ligands was carried out by molecular docking. CpAAT1 structure showed two domains connected by a large crossover loop, with a solvent channel in the centre of the structure. CpAAT1 and VpAAT1 proteins showed similar 3D structures, including their catalytic sites, but their solvent channels showed differences in size and shape. CpAAT1 solvent channel is larger, in agreement with its higher selectivity for large acyl-CoA substrates. In addition, the most favourably predicted substrate orientation in CpAAT1 was observed for methanol and butanoyl-CoA, showing a perfect coincidence with the high production rate of methyl butanoate of C. papaya fruit.     

Computational analysis of DNA photolyases using digital signal processing methods

Sun light energy is used by plants to trigger their growth and development. However, an increase of UV-B light may lead to DNA damage. DNA photolyases are enzymes that repair the cyclobutane pyridine dimer (CPD) and 6–4 photoproduct lesions formed through UV irradiation of DNA. Many aspects of the repair process are under intense scientific investigation but still poorly understood. Here we have computationally analysed DNA-photolyases using the resonant recognition model (RRM), a physico-mathematical approach based on digital signal processing methods. The RRM proposes that protein interactions represent the transfer of resonant electromagnetic energy between interacting molecules at the particular frequency. Within this study we have determined photolyases characteristic frequency, “hot spots” amino acids corresponding to the functional mutations and functional active/binding sites, and designed photolyase peptide analogous. A mutual relationship between photolyase and p53 tumour suppressor protein has also been investigated. The results obtained provide new insights into the structure–function relationships of photolyase protein family.     

Hydrolases: The Correlation Between Informational Structure and the Catalytic Sites Organization

Abstract

The novel method allowing identification of protein structure elements responsible for catalytic activity manifestation is proposed. Structural organization of various hydrolases was studied using the ANIS (ANalysis of Informational Structure) method. ANIS allows to reveal a hierarchy of the ELements of Information Structure (ELIS) using protein amino acid sequence. The ELIS corresponds to the variable length sites with an increased density of structural information. The amino acid residues forming the enzyme catalytic site were shown to belong to the different top-ranking ELIS located in the contact area of the corresponding spatial structure clusters. In the protein spatial structure catalytic sites are located in the area of contact between fragments of polypeptide chain (structural blocs) allocation to the different top-ranking ELIS. According to our results we concluded that structural blocks corresponding to top-ranking ELIS are crucial for protein functioning. Such regions are structurally independent, and their determinate mobility relative to each other is vital for an efficient enzymatic reaction to occur.     

Relationship Between Curved DNA Conformations and Slow Gel Migration

Abstract

We propose some specific DNA conformations that explain, in terms of molecular conformations, the anomalous gel electrophoretic behavior of the sequences (VA₄T₄X)₁, and (V₂A₃X₂)₁ where V and X are either G or C. Previously (J. Biomole. Struct. Dyn. 4, 41, 1986) we considered hydrophobic interactions a mong aliphatic hydrocarbon groups in A/T sequences. In the sequences (T)_n · (A)_n, the T's are slightly bent to yield structures with tightly stacked methyl groups along one side of the major groove. By folding together the two pairs of stacked methyls on the opposite sides of the major groove, TTAA might yield a relatively sharp bend. On this basis, we show below that the sequences (VT₄A₄X)₁ might form a very tightly coiled super-helix whereas the sequences (VA₄T₄X)₁ form a broad super-helix of radius ~ 120 A for i = 25. The sequence (V₂A₃T₃X₂)₁ forms a slightly smaller radius super-helix. The time of passage through the gel has been taken to be inversely proportional to the smallesuiimension of the molecule. Specifically we are taking the ratio of the apparent molecular weight to the actual molecular weight to be related to the moment of inertia I₁ about the smallest principal axis of the molecular conformation. We find a good fit to the experimental gel mobility data of Hagerman (2) if we assume this ratio to be proportional to (I₁)^1/5.     

Protein Sequence Modules

Abstract

Conserved protein sequence segments are commonly believed to correspond to functional sites in the protein sequence. A novel approach is proposed to profile the changing degree of conservation along the protein sequence, by evaluating the occurrence frequencies of all short oligopeptides of the given sequence in a large proteome database. Thus, a protein sequence conservation profile can be plotted for every protein. The profile indicates where along the sequences the potential functional (conserved) sites are located. The corresponding oligopeptides belonging to the sites are very frequent across many prokaryotic species. Analysis of a representative set of such profiles reveals a common feature of all examined proteins: they consist of sequence modules represented by the peaks of conservation. Typical size of the modules (peak-to-peak distance) is 25–30 amino acid residues.     

Conformations of Higher Gangliosides and Their Binding with Cholera Toxin—Investigation by Molecular Modeling,Molecular Mechanics,and Molecular Dynamics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface higher gangliosides (GT1A and GT1B) and their interaction with Cholera Toxin. The water mediated hydrogen bonding network exists between sugar residues in gangliosides. An integrated molecular modeling, molecular mechanics, and molecular dynamics calculation of cholera toxin complexed with GT1A and GT1B reveal that, the active site of cholera toxin can accommodate these higher gangliosides. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different higher ganglioside towards cholera toxin. This study identifies that the binding site of cholera toxin is shallow and can accommodate a maximum of two NeuNAc residues. The NeuNAc binding site of cholera toxin may be crucial for the design of inhibitors that can prevent the infection of cholera.     

3D-QSAR with CoMFA Model of Prolylendopeptidase Substrates

Abstract

The prolylendopeptidase (PEP) is the proteolytic enzyme, which plays an essential role in the regulation of some processes in central nervous system, such as memory, learning and behavior. It was shown that PEP activity changes at different diseases, like Parkinsons or Alzheimer's diseases, and some PEP inhibitors are used in therapy. At present time the discovery of new types of PEP inhibitors are the actual task.

In this study the structure of PEP active site was analyzed by 3D-QSAR with CoMFA methods using of 12 PEP substrates. The designed pharmacophore model assumes that substrates interact with PEP active site by pyrrolidol ring of proline residue and by hydrogen bonding.

The 3-D-QSAR + CoMFA model of PEP substrates propose that the hydrophobic bonds play the essential role in substrate interaction with enzyme. This model reveals the important steric and electrostatic areas around the molecules and the presence of substituents controls the PEP activity for substrates. Analysis of obtained data allows to assume, that substrate binding in PEP active site causes essential perturbations of substrate structure. This effect mainly depends on chemical nature of the amino acid side chain, located near to proline.     

9. LIST OF MEMBERS (as at date of publication)

We studied the breeding ecology of the Chestnut-backed Sparrowlark Eremopterix leucotis over three years between 2008 and 2010. The breeding season was bimodal with a main peak in laying in autumn (March–April) and another smaller peak in spring (September–October). Nest microhabitat analyses showed they prefer nesting in open areas with lots of bare ground (median 67.5%). Nest entrance directions were biased towards the south (mean vector (µ) = 186.44°). The majority of nests (78.2%) had an apron at the nest entrance. The mean clutch size was 1.88 but there was geographic variation in clutch size between northern and southern races of the species. The mean incubation and nestling periods were 10.33 d (range 10–11 d) and 9.20 d (range 8–10 d), respectively. The results suggest that parental contributions during incubation are almost equal, but females made significantly more food deliveries during the nestling period compared to males. The diet of nestlings comprised mainly of invertebrates (50.2%), seeds (34.4%) and unidentified food items (15.4%). Breeding success was low, averaging 16.1% (range 8.1–20.6%), and the average number of fledged young per pair was 0.36 ± 0.71. Replacement broods were common and we also recorded repeat brooding attempts.