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ItemA Threading-Based Method for the Prediction of DNABinding Proteins with Application to the Human GenomeProteins with Application to the Human Genome(Georgia Institute of Technology, 2009-11-13) Gao, Mu ; Skolnick, JeffreyDiverse mechanisms for DNA-protein recognition have been elucidated in numerous atomic complex structures from various protein families. These structural data provide an invaluable knowledge base not only for understanding DNA protein interactions, but also for developing specialized methods that predict the DNA-binding function from protein structure. While such methods are useful, a major limitation is that they require an experimental structure of the target as input. To overcome this obstacle, we develop a threading-based method, DNA-Binding-Domain-Threader (DBD-Threader, for the prediction of DNA-binding domains and associated DNA-binding protein residues. Our method, which uses a template library composed of DNA-protein complex structures, requires only the target protein’s sequence. In our approach,fold similarity and DNA-binding propensity are employed as two functional discriminating properties. In benchmark tests on 179 DNA-binding and 3,797 non-DNA-binding proteins, using templates whose sequence identity is less than 30% to the target, DBD-Threader achieves a sensitivity/precision of 56%/86%. This performance is considerably better than the standard sequence comparison method PSI-BLAST and is comparable to DBD-Hunter, which requires an experimental structure as input. Moreover, for over 70% of predicted DNA-binding domains, the backbone Root Mean Square Deviations (RMSDs) of the top-ranked structural models are within 6.5 A°of their experimental structures, with their associated DNA binding sites identified at satisfactory accuracy. Additionally, DBD-Threader correctly assigned the SCOP superfamily for most predicted domains. To demonstrate that DBD-Threader is useful for automatic function annotation on a large-scale, DBD-Threader was applied to 18,631 protein sequences from the human genome; 1,654 proteins are predicted to have DNA-binding function. Comparison with existing Gene Ontology (GO) annotations suggests that ,30% of our predictions are new. Finally, we present some interesting predictions in detail. In particular, it is estimated that 20% of classic zinc finger domains play a functional role not related to direct DNA-binding.
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ItemFrom Nonspecific DNA–Protein Encounter Complexes to the Prediction of DNA–Protein Interactions(Georgia Institute of Technology, 2009-04-03) Gao, Mu ; Skolnick, JeffreyDNA–protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA–protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA–protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA–protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA–protein interaction modes exhibit some similarity to specific DNA–protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Ca deviation from native is up to 5 Å from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA–protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.
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ItemDBD-Hunter: a knowledge-based method for the prediction of DNA protein interactions(Georgia Institute of Technology, 2008-05-31) Gao, Mu ; Skolnick, JeffreyThe 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.
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ItemOnset of anthrax toxin pore formation(Georgia Institute of Technology, 2006-05) Gao, Mu ; Schulten, KlausProtective antigen (PA) is the anthrax toxin protein recognized by capillary morphogenesis gene 2 (CMG2), a transmembrane cellular receptor. Upon activation, seven ligand-receptor units self-assemble into a heptameric ring-like complex that becomes endocytozed by the host cell. A critical step in the subsequent intoxication process is the formation and insertion of a pore into the endosome membrane by PA. The pore conversion requires a change in binding between PA and its receptor in the acidified endosome environment. Molecular dynamics simulations totaling ;136 ns on systems of over 92,000 atoms were performed. The simulations revealed how the PA-CMG2 complex, stable at neutral conditions, becomes transformed at low pH upon protonation of His-121 and Glu-122, two conserved amino acids of the receptor. The protonation disrupts a salt bridge important for the binding stability and leads to the detachment of PA domain II, which weakens the stability of the PA-CMG2 complex significantly, and subsequently releases a PA segment needed for pore formation. The simulations also explain the great strength of the PA-CMG2 complex achieves through extraordinary coordination of a divalent cation.
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ItemSteered molecular dynamics studies of titin I1 domain unfolding(Georgia Institute of Technology, 2002-12) Gao, Mu ; Wilmanns, Matthias ; Schulten, KlausThe cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the only structurally known Ig domain from the distal region of the titin I-band. In this paper we report SMD simulations unfolding I1, the first structurally available Ig domain from the proximal region of the titin I-band. The simulations are carried out with a view toward upcoming atomic force microscopy experiments. Both constant velocity and constant force stretching have been employed to model mechanical unfolding of oxidized I1, which has a disulfide bond bridging -strands C and E, as well as reduced I1, in which the disulfide bridge is absent. The simulations reveal that I1 is protected against external stress mainly through six interstrand hydrogen bonds between its A and B -strands. The disulfide bond enhances the mechanical stability of oxidized I1 domains by restricting the rupture of backbone hydrogen bonds between the A - and G-strands. The disulfide bond also limits the maximum extension of I1 to 220 Å. Comparison of the unfolding pathways of I1 and I27 are provided and implications to AFM experiments are discussed.
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ItemSimulated refolding of stretched titin immunoglobulin domains(Georgia Institute of Technology, 2001-10) Gao, Mu ; Lu, Hui ; Schulten, KlausSteered molecular dynamics (SMD) is used to investigate forced unfolding and spontaneous refolding of immunoglobulin I27, a domain of the muscle protein titin. Previous SMD simulations revealed the events leading to stretch-induced unfolding of I27, the rupture of hydrogen bonds bridging b-strands A and B, and those bridging b-strands A9 and G, the latter rupture occurring at an extension of ;15 Å and preceding the complete unfolding. Simulations are now used to study the refolding of partially unfolded I27 domains. The results reveal that stretched domains with ruptured interstrand hydrogen bonds shrink along the extension direction. Two types of refolding patterns are recognized: for separated b-strands A9 and G, in most simulations five of the six hydrogen bonds between A9 and G stably reformed in 2 ns, whereas for separated b-strands A and B hydrogen bonds seldom reformed in eight 2-ns simulations. The mechanical stability of the partially refolded intermediates has been tested by re-stretching.