Combining protein evolution and secondary structure

An evolutionary model that combines protein secondary structure and amino acid replacement is introduced. It allows likelihood analysis of aligned protein sequences and does not require the underlying secondary (or tertiary) structures of these sequences to be known. One component of the model describes the organization of secondary structure along a protein sequence and another specifies the evolutionary process for each category of secondary structure. A database of proteins with known secondary structures is used to estimate model parameters representing these two components. Phylogeny, the third component of the model, can be estimated from the data set of interest. As an example, we employ our model to analyze a set of sucrose synthase sequences. For the evolution of sucrose synthase, a parametric bootstrap approach indicates that our model is statistically preferable to one that ignores secondary structure.      

Prediction of 3-D structure of the Cro protein from phage 434

A comparative model building process has been utilized to predict the three-dimensional structure of the bacteriophage 434 Cro protein. Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model. From this model the interactions between the 434 Cro protein and its operator DNA have been deduced. These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.     

Prediction of the tertiary structure of parathyroid-hormone-related protein (residues 1–34) by the island model

Prediction of the tertiary structure of a 34 residue N-terminal fragment of parathyroid-hormone-related protein was carried out by the island model. This peptide is known as a major causative agent of humoral hypercalcemia of malignancy, but structural information studied by X-ray diffraction has not been reported. We adopted the secondary structure determined by NMR and packed on the basis of island model of protein folding developed by us. Predicted structure is discussed in connection with the interaction of active sites.     

Three-dimensional model of the α-subunit of bacterial luciferase

The predicted secondary structure of both subunits of bacterial luciferase is in accordance with a regular 8-fold α/β-barrel structure. The 3D profile^1,2 confirmed that luciferase subunits are compatible with the α/β-barrel despite the absence of sequence similarity with any α/β-barrel protein. The three-dimensional structure of 260 residues of the α-chain of luciferase was modeled from coordinates of glycolate oxidase and then energy minimized. The model obtained satisfies the criteria for the structure of a globular protein and is in accordance with known experimental data. From the model it is possible to predict active site residues involved in binding and catalysis. These predictions, and thus also the model, can be tested by protein engineering experiments. © 1995 Wiley-Liss, Inc.     

Prediction of 3-D Structure of the Cro Protein from Phage 434

Abstract

A comparative model building process has been utilized to predict (he three-dimensional structure of the bacteriophage 434 Cro protein, Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model. From this model the interactions between the 434 Cro protein and its operator DNA have been deduced. These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.     

A structural model for the rolA protein and its interaction with DNA

The study of the plant oncogene rolA has been hampered by a lack of structural information. Here we show that, despite a lack of significant sequence similarity to proteins of known structure, the rolA sequence adopts a known fold; that of the papillomavirus E2 DNA-binding domain. This fold is reliably identified by modern threading programs, which consider predicted secondary structure, but not by others. Although the rolA sequence is only around 16% identical to those of the available template structures, a structural model could be built that performed well against protein structure verification programs. The adopted strategy involved alignment corrections, justified by multiple model building and evaluation, with particular attention paid to the hydrophobic core residues. We find that rolA protein is predicted to resemble the template proteins in two key aspects; existence as a dimer and ability to bind DNA. rolA protein has recently been shown experimentally to possess DNA binding ability. This model predicts Lys 24 and Arg 27 to be involved in sequence-specific interactions and eight other residues to hydrogen-bond phosphate groups of the DNA.     

Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA.

Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another. The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model). The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket. Some minor alterations were made to the rRNA model to improve the fit. In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied. The quality of the fit gives added confidence as to the validity of the 16S rRNA model. Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.     

Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

Distance-dependent atomic knowledge-based force in protein fold recognition

We have recently introduced a novel model for discriminating the correctly folded proteins from well-designed decoy structures using mechanical interatomic forces. In the model, we considered a protein as a collection of springs and the force imposed to each atom was calculated by using the relation between the potential energy and the force. A mean force potential function is obtained from statistical contact preferences within the known protein structures. In this article, the interatomic forces are calculated by numerical derivation of the potential function. For assessing the knowledge-based force function we consider an optimal structure and define a score function on the 3D structure of a protein. We compare the force imposed to each atom of a protein with the corresponding atom in the optimum structure. Afterwards we assign larger scores to those atoms with the lower forces. The total score is the sum of partial scores of atoms. The optimal structure is assumed to be the one with the highest score in the dataset. Finally, several decoy sets are applied in order to evaluate the performance of our model.     

Database of homology-derived protein structures and the structural meaning of sequence alignment

The database of known protein three-dimensional structures can be significantly increased by the use of sequence homology, based on the following observations. (1) The database of known sequences, currently at more than 12,000 proteins, is two orders of magnitude larger than the database of known structures. (2) The currently most powerful method of predicting protein structures is model building by homology. (3) Structural homology can be inferred from the level of sequence similarity. (4) The threshold of sequence similarity sufficient for structural homology depends strongly on the length of the alignment. Here, we first quantify the relation between sequence similarity, structure similarity, and alignment length by an exhaustive survey of alignments between proteins of known structure and report a homology threshold curve as a function of alignment length. We then produce a database of homology-derived secondary structure of proteins (HSSP) by aligning to each protein of known structure all sequences deemed homologous on the basis of the threshold curve. For each known protein structure, the derived database contains the aligned sequences, secondary structure, sequence variability, and sequence profile. Tertiary structures of the aligned sequences are implied, but not modeled explicitly. The database effectively increases the number of known protein structures by a factor of five to more than 1800. The results may be useful in assessing the structural significance of matches in sequence database searches, in deriving preferences and patterns for structure prediction, in elucidating the structural role of conserved residues, and in modeling three-dimensional detail by homology.     

Three-dimensional model for stellacyanin, a "blue" copper-protein.

A three-dimensional model of the "blue" copper-glycoprotein stellacyanin from Rhus vernicifera has been derived by computer graphics, energy minimization and molecular dynamics techniques. The initial atomic co-ordinates were obtained by making substitutions and insertions in the known structure of another blue copper-protein, cucumber basic protein (CBP), which is 46% homologous with stellacyanin and has similar spectroscopic properties. An important difference between CBP and stellacyanin is that the latter lacks methionine, a residue that forms an exceptionally long bond to the copper atom in all blue copper-proteins of known structure. In the aligned amino acid sequences, stellacyanin has glutamine 97 at the position that corresponds to the copper-binding methionine 89 in CBP. The hypothesis that the copper atom in stellacyanin is co-ordinated by the side-chain functional groups of histidine 46, cysteine 87, histidine 92 and glutamine 97 leads to a model that enables the spectroscopic properties, redox potential and electron-transfer kinetics of the protein to be rationalized. The present model for stellacyanin is more plausible than an antecedent model derived from the structure of plastocyanin. This demonstrates that the output from molecular modeling calculations is strongly dependent on the input, and that sequence homology with the target molecule is an important criterion for the selection of a starting model.     

Reconstruction of 3D structures from protein contact maps

The prediction of the protein tertiary structure from solely its residue sequence (the so called Protein Folding Problem) is one of the most challenging problems in Structural Bioinformatics. We focus on the protein residue contact map. When this map is assigned it is possible to reconstruct the 3D structure of the protein backbone. The general problem of recovering a set of 3D coordinates consistent with some given contact map is known as a unit-disk-graph realization problem and it has been recently proven to be NP-Hard. In this paper we describe a heuristic method (COMAR) that is able to reconstruct with an unprecedented rate (3-15 seconds) a 3D model that exactly matches the target contact map of a protein. Working with a non-redundant set of 1760 proteins, we find that the scoring efficiency of finding a 3D model very close to the protein native structure depends on the threshold value adopted to compute the protein residue contact map. Contact maps whose threshold values range from 10 to 18 Ångstroms allow reconstructing 3D models that are very similar to the proteins native structure.     

A computer model to dynamically simulate protein folding: studies with crambin

The current work describes a simplified representation of protein structure with uses in the simulation of protein folding. The model assumes that a protein can be represented by a freely rotating rigid chain with a single atom approximating the effect of each side chain. Potentials describing the attraction or repulsion between different types of amino acids are determined directly from the distribution of amino acids in the database of known protein structures. The optimization technique of simulated annealing has been used to dynamically sample the conformations available to this simple model, allowing the protein to evolve from an extended, random coil into a compact globular structure. Many characteristics expected of true proteins, such as the sequence-dependent formation of secondary structure, the partitioning of hydrophobic residues, and specific disulfide pairing, are reproduced by the simulation, suggesting the model may accurately simulate the folding process.     

What is the protein design alphabet?

Selecting a protein sequence that corresponds to a specific three-dimensional protein structure is known as the protein design problem. One principal bottleneck in solving this problem is our lack of knowledge of precise atomic interactions. Using a simple model of amino acid interactions, we determine three crucial factors that are important for solving the protein design problem. Among these factors is the protein alphabet-a set of sequence elements that encodes protein structure. Our model predicts that alphabet size is independent of protein length, suggesting the possibility of designing a protein of arbitrary length with the natural protein alphabet. We also find that protein alphabet size is governed by protein structural properties and the energetic properties of the protein alphabet units. We discover that the usage of average types of amino acid in proteins is less than expected if amino acids were chosen randomly with naturally occurring frequencies. We propose three possible scenarios that account for amino acid underusage in proteins. These scenarios suggest the possibility that amino acids themselves might not constitute the alphabet of natural proteins.     

An avidin-like domain that does not bind biotin is adopted for oligomerization by the extracellular mosaic protein fibropellin

The protein avidin found in egg white seems optimized for binding the small vitamin biotin as a stable homotetramer. Indeed, along with its streptavidin ortholog in the bacterium Streptomyces avidinii, this protein shows the strongest known noncovalent bond of a protein with a small ligand. A third known member of the avidin family, as similar to avidin as is streptavidin, is found at the C-terminal ends of the multidomain fibropellin proteins found in sea urchin. The fibropellins form a layer known as the apical lamina that surrounds the sea urchin embryo throughout development. Based upon the structure of avidin, we deduced a structural model for the avidin-like domain of the fibropellins and found that computational modeling predicts a lack of biotin binding and the preservation of tetramerization. To test this prediction we expressed and purified the fibropellin avidin-like domain and found it indeed to be a homotetramer incapable of binding biotin. Several lines of evidence suggest that the avidin-like domain causes the entire fibropellin protein to tetramerize. We suggest that the presence of the avidin-like domain serves a structural (tetrameric form) rather than functional (biotin-binding) role and may therefore be a molecular instance of exaptation-the modification of an existing function toward a new function. Finally, based upon the oligomerization of the avidin-like domain, we propose a model for the overall structure of the apical lamina.     

The crystal structure of the superoxide dismutase from Helicobacter pylori reveals a structured C-terminal extension

Superoxide dismutases (SODs) are key enzymes for fighting oxidative stress. Helicobacter pylori produces a single SOD (HpSOD) which contains iron. The structure of this antioxidant protein has been determined at 2.4 A resolution. It is a dimer of two identical subunits with one iron ion per monomer. The protein shares 53% sequence identity with the corresponding enzyme from Escherichia coli. The model is compared with those of other dimeric Fe-containing SODs. HpSOD shows significant differences in relation to other SODs, the most important being an extended C-terminal tail. This structure provides a model for closely related sequences from species such as Campylobacter, where no structures are currently known. The structure of extended carboxyl termini is discussed in light of putative functions it may serve.     

Construction of new ligand binding sites in proteins of known structure. I. Computer-aided modeling of sites with pre-defined geometry.

We have devised a molecular model building computer program (DEZYMER) which builds new ligand binding sites into a protein of known three-dimensional structure. It alters only the sequence and the side-chain structure of the protein, leaving the protein backbone fold intact by definition. The program searches for a constellation of backbone positions arranged such that if appropriate side-chains were placed there, they would bind the ligand according to a pre-defined geometry of interaction specified by the experimentalist. These binding sites are introduced by the program by taking into account simple rules such as steric hindrance, atomic close-packing and hydrogen bond patterns, which are known to maintain the integrity of a protein structure to a first approximation. A test case is presented in this paper where the copper binding site found in blue-copper proteins such as plastocyanin, azurin and cupredoxin is introduced into Escherichia coli thioredoxin. The model building of one of the solutions found by the program is presented in some detail. The experimental construction and properties of this new protein are described in an accompanying paper. It is hoped that this program provides a general method for the design of ligand binding sites and enzyme active sites, which can then be tested experimentally.     

Local function conservation in sequence and structure space

We assess the variability of protein function in protein sequence and structure space. Various regions in this space exhibit considerable difference in the local conservation of molecular function. We analyze and capture local function conservation by means of logistic curves. Based on this analysis, we propose a method for predicting molecular function of a query protein with known structure but unknown function. The prediction method is rigorously assessed and compared with a previously published function predictor. Furthermore, we apply the method to 500 functionally unannotated PDB structures and discuss selected examples. The proposed approach provides a simple yet consistent statistical model for the complex relations between protein sequence, structure, and function. The GOdot method is available online (http://godot.bioinf.mpi-inf.mpg.de).     

Computer aided screening of natural compounds targeting the E6 protein of HPV using molecular docking

The cancer profile in the Indian state of Uttarakhand reveals that the breast cancer is the most prevalent type of cancers in females followed by cervical and ovarian type. Literature survey shows that the E6 protein of Human Papilloma Virus-16 (HPV-16) is responsible for causing several forms of cancer in human. Therefore, it is of interest to screen HPV-16 E6 target protein with known natural compounds using computer aided molecular modeling and docking tools. The complete structure of E6 is unknown. Hence, the E6 structure model was constructed using different online servers followed by molecular docking of Colchine, Curcumin, Daphnoretin, Ellipticine and Epigallocatechin-3-gallate; five known natural compounds with best E6 protein model predicted by Phyre2 server. The screening exercise shows that Daphnoretin (with binding free energy of -8.3 kcal/mol), a natural compound derived from Wikstroemia indica has the top binding properties. Thus, it is of interest to consider the compound for further validation.     

Conformational changes in bacteriophage P22 scaffolding protein induced by interaction with coat protein

Many prokaryotic and eukaryotic double-stranded DNA viruses use a scaffolding protein to assemble their capsid. Assembly of the double-stranded DNA bacteriophage P22 procapsids requires the interaction of 415 molecules of coat protein and 60-300 molecules of scaffolding protein. Although the 303-amino-acid scaffolding protein is essential for proper assembly of procapsids, little is known about its structure beyond an NMR structure of the extreme C-terminus, which is known to interact with coat protein. Deletion mutagenesis indicates that other regions of scaffolding protein are involved in interactions with coat protein and other capsid proteins. Single-cysteine and double-cysteine variants of scaffolding protein were generated for use in fluorescence resonance energy transfer and cross-linking experiments designed to probe the conformation of scaffolding protein in solution and within procapsids. We showed that the N-terminus and the C-terminus are proximate in solution, and that the middle of the protein is near the N-terminus but not accessible to the C-terminus. In procapsids, the N-terminus was no longer accessible to the C-terminus, indicating that there is a conformational change in scaffolding protein upon assembly. In addition, our data are consistent with a model where scaffolding protein dimers are positioned parallel with one another with the associated C-termini.