In silico study of human aquaporin AQP11 and AQP12 channels.

AQP11 and AQP12 are the most distantly related paralogs of the aquaporin family in human. They share indeed a low sequence similarity with other aquaporins and exhibit a modified N-terminal NPA signature motif. Furthermore, they have an anomalous subcellular localization. The AQP11 and AQP12 biological role remains to be fully clarified and their ability to allow transport of water is still debated. We have built accurate 3D-models for AQP11 and AQP12 and comprehensively compared their sequence and structure to other known aquaporins. In order to investigate whether they appear compatible or not with water permeability, we especially focused on the amino acid composition and electrostatics of their channels, keeping the structure of the low-water efficiency AQP0 as a reference system. Our analysis points out a possible alternative ar/R site and shows that these aquaporins feature unique residues at key pore-lining positions that make the shape, composition and electrostatics of their channel peculiar. Such residues can represent pivotal hints to study and explain the AQP11 and AQP12 biological and molecular function.

Water and CO2 permeability of SsAqpZ, the cyanobacterium Synechococcus sp. PCC7942 aquaporin.

BACKGROUND INFORMATION: Cyanobacteria possess Aquaporin-Z (AqpZ) membrane channels which have been suggested to mediate the water efflux underlying osmostress-inducible gene expression and to be essential for glucose metabolism under photomixotrophic growth. However, preliminary observations suggest that the biophy-sical properties of transport and physiological meaning of AqpZ in such photosynthetic microorganisms are not yet completely assessed. RESULTS: In this study, we used Xenopus laevis oocyte and proteoliposome systems to directly demonstrate the water permeability of the cyanobacterium Synechococcus sp. PCC7942 aquaporin, SsAqpZ. By an in vitro assay of intracellular acidification in yeast cells, SsAqpZ was found to transport also CO2 . Consistent with this result, during the entire exponential phase of growth, Synechococcus SsAqpZ-null-mutant cells grew slower than the corresponding wild-type cells. This phenotype was stronger with higher levels of extracellular CO2 . In line with the conversion of CO2 gas into HCO3(-) ions under alkaline conditions, the impairment in growth of the SsAqpZ-null strain was weaker in more alkaline culture medium. CONCLUSIONS: Cyanobacterial SsAqpZ may exert a pleiotropic function in addition to the already reported roles in macronutrient homeostasis and osmotic-stress response as it appears to constitute an important pathway in CO2 uptake, a fundamental step in photosynthesis.

Extending CATH: increasing coverage of the protein structure universe and linking structure with function.

CATH version 3.3 (class, architecture, topology, homology) contains 128,688 domains, 2386 homologous superfamilies and 1233 fold groups, and reflects a major focus on classifying structural genomics (SG) structures and transmembrane proteins, both of which are likely to add structural novelty to the database and therefore increase the coverage of protein fold space within CATH. For CATH version 3.4 we have significantly improved the presentation of sequence information and associated functional information for CATH superfamilies. The CATH superfamily pages now reflect both the functional and structural diversity within the superfamily and include structural alignments of close and distant relatives within the superfamily, annotated with functional information and details of conserved residues. A significantly more efficient search function for CATH has been established by implementing the search server Solr (http://lucene.apache.org/solr/). The CATH v3.4 webpages have been built using the Catalyst web framework.

Mutations at key pore-lining positions differentiate the water permeability of fish lens aquaporin from other vertebrates.

Aquaporin-0 (AQP0) is the major integral membrane protein of lens fiber cell and helps to maintain lens transparency by mediating inter-cell adhesion. To shed light on the unexpected higher water transport efficiency of killifish AQP0 as compared to mammalian orthologues, we performed a comparative analysis of all available AQP0 sequences and built 3D-models for representatives of different vertebrate classes. The analysis shows that air-living organisms evolved specific mutations at pore-lining positions to modulate the AQP0 water transport efficiency while maintaining the correct tertiary/quaternary arrangement to allow the formation of "thin junctions" between lens fiber cells. We conclude that the low permeability of mammalian AQP0 is required not to promote cell adhesion, but to modulate the water balance in a dry environment.

Electrostatics of aquaporin and aquaglyceroporin channels correlates with their transport selectivity.

Aquaporins are homotetrameric channel proteins, which allow the diffusion of water and small solutes across biological membranes. According to their transport function, aquaporins can be divided into "orthodox aquaporins", which allow the flux of water molecules only, and "aquaglyceroporins", which facilitate the diffusion of glycerol and other small solutes in addition to water. The contribution of individual residues in the pore to the selectivity of orthodox aquaporins and aquaglyceroporins is not yet fully understood. To gain insights into aquaporin selectivity, we focused on the sequence variation and electrostatics of their channels. The continuum Poisson-Boltzmann electrostatic potential along the channel was calculated and compared for ten three-dimensional-structures which are representatives of different aquaporin subfamilies, and a panel of functionally characterized mutants, for which high-accuracy three-dimensional-models could be derived. Interestingly, specific electrostatic profiles associated with the main selectivity to water or glycerol could be identified. In particular: (i) orthodox aquaporins showed a distinctive electrostatic potential maximum at the periplasmic side of the channel around the aromatic/Arg (ar/R) constriction site; (ii) aquaporin-0 (AQP0), a mammalian aquaporin with considerably low water permeability, had an additional deep minimum at the cytoplasmic side; (iii) aquaglyceroporins showed a rather flat potential all along the channel; and (iv) the bifunctional protozoan PfAQP had an unusual all negative profile. Evaluation of electrostatics of the mutants, along with a thorough sequence analysis of the aquaporin pore-lining residues, illuminated the contribution of specific residues to the electrostatics of the channels and possibly to their selectivity.

PoreLogo: a new tool to analyse, visualize and compare channels in transmembrane proteins.

UNLABELLED: The increasing number of available atomic 3D structures of transmembrane channel proteins represents a valuable resource for better understanding their structure-function relationships and to eventually predict their selectivity. Herein, we present PoreLogo, an automatic tool for analysing, visualizing and comparing the amino acid composition of transmembrane channels and its conservation across the corresponding protein family. AVAILABILITY: PoreLogo is accessible as a public web server at http://www.ebi.ac.uk/thornton-srv/software/PoreLogo/.

PoreWalker: a novel tool for the identification and characterization of channels in transmembrane proteins from their three-dimensional structure.

Transmembrane channel proteins play pivotal roles in maintaining the homeostasis and responsiveness of cells and the cross-membrane electrochemical gradient by mediating the transport of ions and molecules through biological membranes. Therefore, computational methods which, given a set of 3D coordinates, can automatically identify and describe channels in transmembrane proteins are key tools to provide insights into how they function.Herein we present PoreWalker, a fully automated method, which detects and fully characterises channels in transmembrane proteins from their 3D structures. A stepwise procedure is followed in which the pore centre and pore axis are first identified and optimised using geometric criteria, and then the biggest and longest cavity through the channel is detected. Finally, pore features, including diameter profiles, pore-lining residues, size, shape and regularity of the pore are calculated, providing a quantitative and visual characterization of the channel. To illustrate the use of this tool, the method was applied to several structures of transmembrane channel proteins and was able to identify shape/size/residue features representative of specific channel families. The software is available as a web-based resource at http://www.ebi.ac.uk/thornton-srv/software/PoreWalker/.

Structure and mechanism of drug efflux machinery in Gram negative bacteria.

In Gram-negative bacteria, multi-component machines that span the inner and outer membranes actively extrude drugs and other toxic small compounds. Many of these machines are assembled principally from three different types of components: i) an outer membrane protein that acts as a channel and opens from a sealed resting state during the transport process, ii) an inner membrane protein that transduces proton electrochemical energy into vectorial displacement of the transported compounds, and iii) a bridging, periplasmic component that links the inner and outer membrane proteins. The pumps may assemble transiently, and the association of components is favoured by engaged substrate and the trans-membrane electrochemical potential. We describe recent structural and functional studies on the individual pump components and discuss models that explain how they associate in the dynamic, active assembly. Based on the available data, we suggest that the assembly of these multi-drug efflux pumps is accompanied by induced fit of the outer membrane component driven mainly by accommodation of the periplasmic component.

Revisiting the prediction of protein function at CASP6.

The ability to predict the function of a protein, given its sequence and/or 3D structure, is an essential requirement for exploiting the wealth of data made available by genomics and structural genomics projects and is therefore raising increasing interest in the computational biology community. To foster developments in the area as well as to establish the state of the art of present methods, a function prediction category was tentatively introduced in the 6th edition of the Critical Assessment of Techniques for Protein Structure Prediction (CASP) worldwide experiment. The assessment of the performance of the methods was made difficult by at least two factors: (a) the experimentally determined function of the targets was not available at the time of assessment; (b) the experiment is run blindly, preventing verification of whether the convergence of different predictions towards the same functional annotation was due to the similarity of the methods or to a genuine signal detectable by different methodologies. In this work, we collected information about the methods used by the various predictors and revisited the results of the experiment by verifying how often and in which cases a convergent prediction was obtained by methods based on different rationale. We propose a method for classifying the type and redundancy of the methods. We also analyzed the cases in which a function for the target protein has become available. Our results show that predictions derived from a consensus of different methods can reach an accuracy as high as 80%. It follows that some of the predictions submitted to CASP6, once reanalyzed taking into account the type of converging methods, can provide very useful information to researchers interested in the function of the target proteins.

Towards genome-scale structure prediction for transmembrane proteins.

In this paper we briefly review some of the recent progress made by ourselves and others in developing methods for predicting the structures of transmembrane proteins from amino acid sequence. Transmembrane proteins are an important class of proteins involved in many diverse biological functions, many of which have great impact in terms of disease mechanism and drug discovery. Despite their biological importance, it has proven very difficult to solve the structures of these proteins by experimental techniques, and so there is a great deal of pressure to develop effective methods for predicting their structure. The methods we discuss range from methods for transmembrane topology prediction to new methods for low resolution folding simulations in a knowledge-based force field. This potential is designed to reproduce the properties of the lipid bilayer. Our eventual aim is to apply these methods in tandem so that useful three-dimensional models can be built for a large fraction of the transmembrane protein domains in whole proteomes.

Identification of a novel putative mitogen-activated kinase cascade on human chromosome 21 by computational approaches.

UNLABELLED: Down syndrome (DS) is the most frequent form of mental retardation and is caused by chromosome 21 (HSA21) trisomy. Despite the number of known genes involved in DS and its high therapeutic interest, biological mechanisms leading to the DS phenotype are not fully clear. We present a functional hypothesis based on fold recognition and hidden Markov model techniques for four HSA21 genes located in the DS Candidate Region (DSCR). More specifically, we propose that they are members of a novel mitogen-activated protein kinase pathway with DYRK1A, SNF1LK and RIPK4 gene products being elements of the kinase cascade and the DSCR3 acting as structural scaffold for their interaction. This hypothesis finds support in various biochemical studies concerning the biological behavior and features of the involved HSA21 proteins. Our analysis calls for specifically designed experiments to validate our prediction and establish its relevance in terms of therapeutic approaches to the disease. CONTACT: anna.tramontano@uniroma1.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Detecting DNA-binding helix-turn-helix structural motifs using sequence and structure information.

In this work, we analyse the potential for using structural knowledge to improve the detection of the DNA-binding helix-turn-helix (HTH) motif from sequence. Starting from a set of DNA-binding protein structures that include a functional HTH motif and have no apparent sequence similarity to each other, two different libraries of hidden Markov models (HMMs) were built. One library included sequence models of whole DNA-binding domains, which incorporate the HTH motif, the second library included shorter models of 'partial' domains, representing only the fraction of the domain that corresponds to the functionally relevant HTH motif itself. The libraries were scanned against a dataset of protein sequences, some containing the HTH motifs, others not. HMM predictions were compared with the results obtained from a previously published structure-based method and subsequently combined with it. The combined method proved more effective than either of the single-featured approaches, showing that information carried by motif sequences and motif structures are to some extent complementary and can successfully be used together for the detection of DNA-binding HTHs in proteins of unknown function.

Neuronal nicotinic acetylcholine receptor agonists: pharmacophores, evolutionary QSAR and 3D-QSAR models.

Neuronal nicotinic acetylcholine ion channel receptors (nAChRs) exist as several subtypes and are involved in a variety of functions and disorders of the central nervous system (CNS), such as Alzheimer's and Parkinson's diseases. The lack of reliable information on the 3D structure of nAChRs prompted us to focus efforts on pharmacophore and structure-affinity relationships (SAFIRs). The use of DISCO (DIStance COmparison) and Catalyst/HipHop led to the formulation of a pharmacophore that is made of three geometrically unrelated features: (i) an ammonium head involved in coulombic and/or H-bond interactions, (ii) a lone pair of a pyridine nitrogen or a carbonyl oxygen, as H-bond acceptor site, and (iii) a hydrophobic molecular region generally constituted by aliphatic cycles. The quantitative SAFIR (QSAFIR) study was carried out on about three hundred nicotinoid agonists, and coherent results were obtained from classical Hansch-type approach, 3D QSAFIRs, based on Comparative Molecular Field Analysis (CoMFA), and trade-off models generated by Multi-objective Genetic QSAR (MoQSAR), a novel evolutionary software that makes use of Genetic Programming (GP) and multi-objective optimization (MO). Within each congeneric series, Hansch-type equations revealed detrimental steric effects as the major factors modulating the receptor affinity, whereas CoMFA allowed us to merge progressively single-class models in a more global one, whose robustness was supported by crossvalidation, high prediction statistics and satisfactory predictions of the affinity data of a true external ligand set (r(2)(pred) = 0.796). Next, MoQSAR was used to analyze a data set of 58 highly active nicotinoids characterized by 56 descriptors, that are log P, MR and 54 low inter-correlated WHIM (Weighted Holistic Invariant Molecular) indices. Equivalent QSAFIR models, that represent different compromises between structural model complexity, fitting and internal model complexity, were found. Our attention was mostly engaged by a number of nonlinear QSAFIRs, which relate nAChR affinity with the log P and directional WHIM descriptors. The results reviewed herein show as QSAFIRs may helpfully complement the pharmacophores, thus enhancing the applicability of computer-aided methodologies in the field of nAChR agonists.