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Center for the Study of Systems Biology

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School of Biological Sciences

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Fr-TM-align: a new protein structural alignment method based on fragment alignments and the TM-score

(Georgia Institute of Technology, 2008-12-12) Pandit, Shashi Bhushan ; Skolnick, Jeffrey

Background: Protein tertiary structure comparisons are employed in various fields of contemporary structural biology. Most structure comparison methods involve generation of an initial seed alignment, which is extended and/or refined to provide the best structural superposition between a pair of protein structures as assessed by a structure comparison metric. One such metric, the TM-score, was recently introduced to provide a combined structure quality measure of the coordinate root mean square deviation between a pair of structures and coverage. Using the TM-score, the TM-align structure alignment algorithm was developed that was often found to have better accuracy and coverage than the most commonly used structural alignment programs; however, there were a number of situations when this was not true. Results: To further improve structure alignment quality, the Fr-TM-align algorithm has been developed where aligned fragment pairs are used to generate the initial seed alignments that are then refined using dynamic programming to maximize the TM-score. For the assessment of the structural alignment quality from Fr-TM-align in comparison to other programs such as CE and TMalign, we examined various alignment quality assessment scores such as PSI and TM-score. The assessment showed that the structural alignment quality from Fr-TM-align is better in comparison to both CE and TM-align. On average, the structural alignments generated using Fr-TM-align have a higher TM-score (~9%) and coverage (~7%) in comparison to those generated by TM-align. Fr- TM-align uses an exhaustive procedure to generate initial seed alignments. Hence, the algorithm is computationally more expensive than TM-align. Conclusion: Fr-TM-align, a new algorithm that employs fragment alignment and assembly provides better structural alignments in comparison to TM-align. The source code and executables of Fr- TM-align are freely downloadable at: http://cssb.biology.gatech.edu/skolnick/files/FrTMalign/.
Benchmarking of TASSER_2.0: an improved protein structure prediction algorithm with more accurate predicted contact restraints

(Georgia Institute of Technology, 2008-08) Lee, Seung Yup ; Skolnick, Jeffrey

To improve tertiary structure predictions of more difficult targets, the next generation of TASSER, TASSER_2.0, has been developed. TASSER_2.0 incorporates more accurate side-chain contact restraint predictions from a new approach, the composite-sequence method, based on consensus restraints generated by an improved threading algorithm, PROSPECTOR_3.5, which uses computationally evolved and wild-type template sequences as input. TASSER_2.0 was tested on a large-scale, benchmark set of 2591 nonhomologous, single domain proteins " 200 residues that cover the Protein Data Bank at 35% pairwise sequence identity. Compared with the average fraction of accurately predicted side-chain contacts of 0.37 using PROSPECTOR_3.5 with wildtype template sequences, the average accuracy of the composite-sequence method increases to 0.60. The resulting TASSER_2.0 models are closerto their native structures, with an average root mean-square deviation of 4.99 A compared to the 5.31 A result of TASSER. Defining a successful prediction as a model with a root mean-square deviation to native < 6.5 A. the success rate of TASSER_2.0 (TASSER) for Medium targets (targets with good templates/poor alignments) is 74.3% (64.7%) and 40.8% (35.5%) for the Hard targets (incorrect templates/alignments). For Easy targets (good templates/alignments), the success rate slightly increases from 86.3% to 88.4%
Identification of metabolites with anticancer properties by computational metabolomics

(Georgia Institute of Technology, 2008-06-17) Arakaki, Adrian K. ; Mezencev, Roman ; Bowen, Nathan J. ; Huang, Ying ; McDonald, John F. ; Skolnick, Jeffrey

Background: Certain endogenous metabolites can influence the rate of cancer cell growth. For example, diacylglycerol, ceramides and sphingosine, NAD+ and arginine exert this effect by acting as signaling molecules, while carrying out other important cellular functions. Metabolites can also be involved in the control of cell proliferation by directly regulating gene expression in ways that are signaling pathway-independent, e.g. by direct activation of transcription factors or by inducing epigenetic processes. The fact that metabolites can affect the cancer process on so many levels suggests that the change in concentration of some metabolites that occurs in cancer cells could have an active role in the progress of the disease. Results: CoMet, a fully automated Computational Metabolomics method to predict changes in metabolite levels in cancer cells compared to normal references has been developed and applied to Jurkat T leukemia cells with the goal of testing the following hypothesis: Up or down regulation in cancer cells of the expression of genes encoding for metabolic enzymes leads to changes in intracellular metabolite concentrations that contribute to disease progression. All nine metabolites predicted to be lowered in Jurkat cells with respect to lymphoblasts that were examined (riboflavin, tryptamine, 3- sulfino-L-alanine, menaquinone, dehydroepiandrosterone, α-hydroxystearic acid, hydroxyacetone, seleno-L-methionine and 5,6-dimethylbenzimidazole), exhibited antiproliferative activity that has not been reported before, while only two (bilirubin and androsterone) of the eleven tested metabolites predicted to be increased or unchanged in Jurkat cells displayed significant antiproliferative activity. Conclusion: These results: a) demonstrate that CoMet is a valuable method to identify potential compounds for experimental validation, b) indicate that cancer cell metabolism may be regulated to reduce the intracellular concentration of certain antiproliferative metabolites, leading to uninhibited cellular growth and c) suggest that many other endogenous metabolites with important roles in carcinogenesis are awaiting discovery.
DBD-Hunter: a knowledge-based method for the prediction of DNA protein interactions

(Georgia Institute of Technology, 2008-05-31) Gao, Mu ; Skolnick, Jeffrey

The structures of DNA–protein complexes have illuminated the diversity of DNA–protein binding mechanisms shown by different protein families. This lack of generality could pose a great challenge for predicting DNA–protein interactions. To address this issue, we have developed a knowledge-based method, DNA-binding Domain Hunter (DBD-Hunter), for identifying DNA-binding proteins and associated binding sites. The method combines structural comparison and the evaluation of a statistical potential, which we derive to describe interactions between DNA base pairs and protein residues. We demonstrate that DBD-Hunter is an accurate method for predicting DNA-binding function of proteins, and that DNA-binding protein residues can be reliably inferred from the corresponding templates if identified. In benchmark tests on ~4000 proteins, our method achieved an accuracy of 98% and a precision of 84%, which significantly outperforms three previous methods. We further validate the method on DNAbinding protein structures determined in DNA-free (apo) state. Weshow that the accuracy of our method is only slightly affected on apo-structures compared to the performance on holo-structures cocrystallized with DNA. Finally, we apply the method to ~1700 structural genomics targets and predict that 37 targets with previously unknown function are likely to be DNA-binding proteins.
The Mosaic Genome of Anaeromyxobacter dehalogenans Strain 2CP-C Suggests an Aerobic Common Ancestor to the Delta-Proteobacteria

(Georgia Institute of Technology, 2008-05-07) Thomas, Sara H. ; Wagner, Ryan D. ; Arakaki, Adrian K. ; Skolnick, Jeffrey ; Kirby, John R. ; Shimkets, Lawrence J. ; Sanford, Robert A. ; Löffler, Frank E.

Anaeromyxobacter dehalogenans strain 2CP-C is a versaphilic delta-Proteobacterium distributed throughout many diverse soil and sediment environments. 16S rRNA gene phylogenetic analysis groups A. dehalogenans together with the myxobacteria, which have distinguishing characteristics including strictly aerobic metabolism, sporulation, fruiting body formation, and surface motility. Analysis of the 5.01 Mb strain 2CP-C genome substantiated that this organism is a myxobacterium but shares genotypic traits with the anaerobic majority of the delta-Proteobacteria (i.e., the Desulfuromonadales). Reflective of its respiratory versatility, strain 2CP-C possesses 68 genes coding for putative c-type cytochromes, including one gene with 40 heme binding motifs. Consistent with its relatedness to the myxobacteria, surface motility was observed in strain 2CP-C and multiple types of motility genes are present, including 28 genes for gliding, adventurous (A-) motility and 17 genes for type IV pilus-based motility (i.e., social (S-) motility) that all have homologs in Myxococcus xanthus. Although A. dehalogenans shares many metabolic traits with the anaerobic majority of the delta- Proteobacteria, strain 2CP-C grows under microaerophilic conditions and possesses detoxification systems for reactive oxygen species. Accordingly, two gene clusters coding for NADH dehydrogenase subunits and two cytochrome oxidase gene clusters in strain 2CP-C are similar to those in M. xanthus. Remarkably, strain 2CP-C possesses a third NADH dehydrogenase gene cluster and a cytochrome cbb3 oxidase gene cluster, apparently acquired through ancient horizontal gene transfer from a strictly anaerobic green sulfur bacterium. The mosaic nature of the A. dehalogenans strain 2CP-C genome suggests that the metabolically versatile, anaerobic members of the delta-Proteobacteria may have descended from aerobic ancestors with complex lifestyles.
Development of a physics-based force field for the scoring and refinement of protein models

(Georgia Institute of Technology, 2008-04) Wróblewska, Liliana ; Jagielska, Anna ; Skolnick, Jeffrey

The minimal requirements of a physics-based potential that can refine protein structures are the existence of a correlation between the energy with native similarity and the scoring of the native structure as the lowest in energy. To develop such a force field, the relative weights of the Amber ff03 all-atom potential supplemented by an explicit hydrogen-bond potential were adjusted by global optimization of energetic and structural criteria for a large set of protein decoys generated for a set of 58 nonhomologous proteins. The average correlation coefficient of the energy with TM-score significantly improved from 0.25 for the original ff03 potential to 0.65 for the optimized force field. The fraction of proteins for which the native structure had lowest energy increased from 0.22 to 0.90. Moreover, use of an explicit hydrogen-bond potential improves scoring performance of the force field. Promising preliminary results were obtained in applying the optimized potentials to refine protein decoys using only an energy criterion to choose the best decoy among sampled structures. For a set of seven proteins, 63% of the decoys improve, 18% get worse, and 19% are not changed.
RNA Enzymes: From Folding to Function in Living Cells

(Georgia Institute of Technology, 2008-03-25) Fedor, Martha J.

Our research aims to generate fundamental insights into catalysis by RNA enzymes and into the pathways through which RNAs form specific functional structures. RNA catalysis remains an intriguing puzzle that has grown in significance since the recent discoveries that the ribosome itself is an RNA enzyme and that human and bacterial mRNAs contain self-cleaving ribozymes. The hairpin ribozyme catalyzes a reversible self-cleavage reaction in which nucleophilic attack of a ribose 2’ hydroxyl on an adjacent phosphorus proceeds through a trigonal bipyramidal trasition state that leads to the formation of 2’,3’-cyclic phosphate and 2’ hydroxyl termini. The metal cation independence of activity and the availability of high-resolution active site structures have made the hairpin ribozyme the prototype for nucleobase-mediated catalytic chemistry. A network of stacking and hydrogen bonding interactions align the reactive phosphate in the appropriate orientation for an S_N2-type nucleophilic attack and orient nucleotide base functional groups near the reactive phosphate to facilitate catalytic chemistry. Two active site nucleobases, G8 and A38, adopt orientations reminiscent of the histidine residues that mediate general acid base catalysis in ribonuclease A, a protein enzyme that catalyzes the same phosphodiester cleavage chemistry. However, our biochemical experiments argue against analogous roles for G8 and A38 in hairpin ribozyme catalysis and suggest that these residues contribute to catalysis through positioning and orientation and electrostatic stabilization of the electronegative transition site. Ribozymes are useful model systems for investigation of RNA folding, since self-cleavage reflects the assembly of a precise functional structure. To learn how structure-function principles revealed through in vitro experiments translate to the behavior of RNA in living cells, we devised a way to evaluate RNA assembly in vivo using RNA self-cleavage rates to quantify assembly of functional RNA structures. Results of these studies show that intracellular RNA folding kinetics and equilibria are indistinguishable from RNA folding behavior in vitro, provided that in vitro folding reactions approximate the ionic conditions characteristic of an intracellular environment. These studies contribute basic knowledge of RNA structure and function and provide a framework for developing technical and therapeutic application involving RNAs as targets and reagents.
New RNA-binding peptidomimetic structures that repress HIV viral replication by specifically inhibiting transcriptional activation

(Georgia Institute of Technology, 2008-03-18) Varani, Gabriele

The Interaction between the human immunodeficiency virus (HIV-1) transactivator protein Tat and its response element TAR plays an essential role in viral replication by controlling HIV transcription. Previous attempts to inhibit this interaction have failed to yield molecules with sufficient potency and specificity to warrant pharmaceutical development. We have shown that comformationally constrained cyclic peptide structural mimics of Tat provide nM inhibitors of the Tat-TAR interaction. These peptidomimetics are proteolytically stable, penetrate cells efficiently and have no cytotoxicity. They specifically inhibit Tat-dependent activation of transcription in cells and repress replication of a wide variety of viral strains representing all the major HIV clades in primary human lymphocytes. The potency and selectivity observed for this family of peptides is unprecedented among Tat inhibitors and suggest that these types of compounds may be widely useful for the pharmacological inhibition of other protein-RNA interactions.
Positive Selection on Non-coding Sequences During Human Evolution: From Genome to Nucleotide

(Georgia Institute of Technology, 2008-03-04) Wray, Gregory
Better living through biosensors

(Georgia Institute of Technology, 2008-02-19) Plaxco, Kevin

The ideal sensor will be sensitive, specific, versatile, small enough to hold in your hand, and selective enough to work even when faced with complex, contaminant-ridden samples. Given the affinity, specificity and generalizability of biomolecular recognition, biosensors have been widely touted for their potential to meet these challenging goals. To date, however, the translation of protein- and DNA-binding events into convenient, highly selective sensing platforms has proven problematic. We have solved this problem by employing the ligand-induced folding of proteins, peptides and DNA as a robust signal transduction mechanism. Our folding-based sensors are rapid (minutes to seconds), sensitive (micromolar to femtomolar), fully electronic, and generalizable to an enormous range of protein, nucleic acid and small molecule targets. The sensors are also reagentless, greater than 99% reusable, and selective enough to be employed in (and re-used from) blood, soil and other grossly contaminated materials. Because of their sensitivity, background suppression, operational convenience and impressive scalability folding-based biosensors appear ideally suited for electronic, on-chip applications in pathogen detection, proteomics and genomics.