FRA1 Kinesin Modulates the Lateral Stability of Cortical Microtubules through Cellulose Synthase-Microtubule Uncoupling Proteins.

Cell wall assembly requires harmonized deposition of cellulose and matrix polysaccharides. Cortical microtubules orient the deposition of cellulose by guiding the trajectory of cellulose synthase complexes. Vesicles containing matrix polysaccharides are thought to be transported by the FRAGILE FIBER1 (FRA1) kinesin to facilitate their secretion along cortical microtubules. The cortical microtubule cytoskeleton thus may provide a platform to coordinate the delivery of cellulose and matrix polysaccharides, but the underlying molecular mechanisms remain unknown. Here, we show that the tail region of the Arabidopsis (Arabidopsis thaliana) FRA1 kinesin physically interacts with cellulose synthase-microtubule uncoupling (CMU) proteins that are important for the microtubule-dependent guidance of cellulose synthase complexes. Interaction with CMUs did not affect microtubule binding or motility of the FRA1 kinesin but differentially affected the protein levels and microtubule localization of CMU1 and CMU2, thus regulating the lateral stability of cortical microtubules. Phosphorylation of the FRA1 tail region inhibited binding to CMUs and consequently reversed the extent of cortical microtubule decoration by CMU1 and CMU2. Genetic experiments demonstrated the significance of this interaction to the growth and reproduction of Arabidopsis plants. We propose that modulation of CMU protein levels and microtubule localization by FRA1 provides a mechanism that stabilizes the sites of deposition of both cellulose and matrix polysaccharides.

Thermal stability and unfolding pathways of Sso7d and its mutant F31A: insight from molecular dynamics simulation.

The thermo-stability and unfolding behaviors of a small hyperthermophilic protein Sso7d as well as its single-point mutation F31A are studied by molecular dynamics simulation at temperatures of 300 K, 371 K and 500 K. Simulations at 300 K show that the F31A mutant displays a much larger flexibility than the wild type, which implies that the mutation obviously decreases the protein's stability. In the simulations at 371 K, although larger fluctuations were observed, both of these two maintain their stable conformations. High temperature simulations at 500 K suggest that the unfolding of these two proteins evolves along different pathways. For the wild-type protein, the C-terminal alpha-helix is melted at the early unfolding stage, whereas it is destroyed much later in the unfolding process of the F31A mutant. The results also show that the mutant unfolds much faster than its parent protein. The deeply buried aromatic cluster in the F31A mutant dissociates quickly relative to the wild-type protein at high temperature. Besides, it is found that the triple-stranded antiparallel ß-sheet in the wild-type protein plays an important role in maintaining the stability of the entire structure.

A holistic molecular docking approach for predicting protein-protein complex structure.

A holistic protein-protein molecular docking approach, HoDock, was established, composed of such steps as binding site prediction, initial complex structure sampling, refined complex structure sampling, structure clustering, scoring and final structure selection. This article explains the detailed steps and applications for CAPRI Target 39. The CAPRI result showed that three predicted binding site residues, A191HIS, B512ARG and B531ARG, were correct, and there were five submitted structures with a high fraction of correct receptor-ligand interface residues, indicating that this docking approach may improve prediction accuracy for protein-protein complex structures.

Protein-protein docking with binding site patch prediction and network-based terms enhanced combinatorial scoring.

Protein-protein docking has made much progress in recent years, but challenges still exist. Here we present the application of our docking approach HoDock in CAPRI. In this approach, a binding site prediction is implemented to reduce docking sampling space and filter out unreasonable docked structures, and a network-based enhanced combinatorial scoring function HPNCscore is used to evaluate the decoys. The experimental information was combined with the predicted binding site to pick out the most likely key binding site residues. We applied the HoDock method in the recent rounds of the CAPRI experiments, and got good results as predictors on targets 39, 40, and 41. We also got good results as scorers on targets 35, 37, 40, and 41. This indicates that our docking approach can contribute to the progress of protein-protein docking methods and to the understanding of the mechanism of protein-protein interactions.

Development of a high-throughput assay for the HIV-1 integrase disintegration reaction.

Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro. In this study, IN and IN-CCD proteins were expressed and purified, and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction. IN exhibited a marked preference for Mn(2+) over Mg(2+) as the divalent cation cofactor in disintegration. Baicalein, a known IN inhibitor, was found to be an IN-CCD inhibitor. The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN, especially targeting IN-CCD.

Molecular dynamics simulation of the transmembrane subunit of BtuCD in the lipid bilayer.

Based on the crystal structure of the vitamin B(12) transporter protein of Escherichia coli (BtuCD) a system consisting of the BtuCD transmembrane domain (BtuC) and the palmitoyloleoyl phosphatidylcholine (POPC) lipid bilayer was constructed in silica, and a more-than-57-nanosecond molecular dynamics (MD) simulation was performed on it to reveal the intrinsic functional motions of BtuC. The results showed that a stable protein-lipid bilayer was obtained and the POPC lipid bilayer was able to adjust its thickness to match the embedded BtuC which underwent relatively complicated motions. These results may help to understand the mechanism of transmembrane substrate transport at the atomic level.

The B73 maize genome: complexity, diversity, and dynamics.

We report an improved draft nucleotide sequence of the 2.3-gigabase genome of maize, an important crop plant and model for biological research. Over 32,000 genes were predicted, of which 99.8% were placed on reference chromosomes. Nearly 85% of the genome is composed of hundreds of families of transposable elements, dispersed nonuniformly across the genome. These were responsible for the capture and amplification of numerous gene fragments and affect the composition, sizes, and positions of centromeres. We also report on the correlation of methylation-poor regions with Mu transposon insertions and recombination, and copy number variants with insertions and/or deletions, as well as how uneven gene losses between duplicated regions were involved in returning an ancient allotetraploid to a genetically diploid state. These analyses inform and set the stage for further investigations to improve our understanding of the domestication and agricultural improvements of maize.

Uncovering the properties of energy-weighted conformation space networks with a hydrophobic-hydrophilic model.

The conformation spaces generated by short hydrophobic-hydrophilic (HP) lattice chains are mapped to conformation space networks (CSNs). The vertices (nodes) of the network are the conformations and the links are the transitions between them. It has been found that these networks have "small-world" properties without considering the interaction energy of the monomers in the chain, i. e. the hydrophobic or hydrophilic amino acids inside the chain. When the weight based on the interaction energy of the monomers in the chain is added to the CSNs, it is found that the weighted networks show the "scale-free" characteristic. In addition, it reveals that there is a connection between the scale-free property of the weighted CSN and the folding dynamics of the chain by investigating the relationship between the scale-free structure of the weighted CSN and the noted parameter Z score. Moreover, the modular (community) structure of weighted CSNs is also studied. These results are helpful to understand the topological properties of the CSN and the underlying free-energy landscapes.

Evolving model of amino acid networks.

The three-dimensional structure of a protein can be treated as a complex network composed of amino acids, and the network properties can help us to understand the relationship between structure and function. Since the amino acid network of a protein is formed in the process of protein folding, it is difficult for general network models to explain its evolving mechanism. Based on the perspective of protein folding, we propose an evolving model for amino acid networks. In our model, the evolution starts from the amino acid sequence of a native protein and it is guided by two generic assumptions: i.e., the neighbor preferential rule and the energy preferential rule. We find that the neighbor preferential rule predominates the general network properties and the energy preferential rule predominates the specific biological structure characteristics. Applied to native proteins, our model mimics the features of amino acid networks well.

Three-dimensional QSAR analyses of 1,3,4-trisubstituted pyrrolidine-based CCR5 receptor inhibitors.

In this study, a series of 1,3,4-trisubstituted pyrrolidine-based CCR5 receptor inhibitors were taken as our target with the method of the three-dimensional quantitative structure-activity relationship (3D-QSAR) analyses in order to investigate the interactions between CCR5 receptor and their inhibitors. For a comparison, Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) were, respectively, used to build predictive models, which were generated from a training set consisting of 72 selected molecules, derived from literatures. Two alignment rules, including rigid body rms (root mean square) fit and field fit, were performed in the superimposition of inhibitors structures. As a result, a better CoMFA model based on common structure alignment obtains a conventional correlation coefficient r(2) of 0.952 and a leave-one-out cross-validated coefficient q(2) of 0.637, while the desirable CoMSIA model based on the same alignment rule acquires the r(2) of 0.958 and the q(2) of 0.677. To further validate the reliability of the models, we also investigated into the externally test set composed of 39 molecules under the criterions of squared correlation coefficient between experimental and predicted activities with intercept R(2) and without intercept R(0)(2), along with R(m)(2) as the modified R(2) with a penalty function due to difference between R(2) and R(0)(2). At last, the contour map also provides a visual representation of contributions of steric, electrostatic, hydrogen bond and hydrophobic fields, as well as the prospective binding modes. These results may provide meaningful guidance to the further work including the similar lead compounds' structure modification and activity prediction.

Study on the mechanism of the BtuF periplasmic-binding protein for vitamin B12.

BtuF is the periplasmic binding protein (PBP) that binds vitamin B(12) and delivers it to the periplasmic surface of the ABC transporter BtuCD. PBPs generally exhibit considerable conformational changes during ligand binding process, however, BtuF belongs to a subclass of PBPs that, doesn't show such behavior on the basis of the crystal structures. Employing steered molecular dynamics on the B(12)-bound BtuF, we investigated the energetics and mechanism of BtuF. A potential of mean force along the postulated vitamin B(12) unbinding pathway was constructed through Jarzynski's equality. The large free energy differences of the postulated B(12) unbinding process suggests the B(12)-bound structure is in a stable closed state and some conformation changes may be necessary to the B(12) unbinding. From the result of the principal component analysis, we found the BtuF-B(12) complex shows clear opening-closing and twisting motion tendencies which may facilitate the unbinding of B(12) from the binding pocket. The intrinsic flexibility of BtuF was also explored, and it's suggested the Trp44-Gln45 pair, which is situated at the mouth of the B(12) binding pocket, may act as a gate in the B(12) binding and unbinding process.

Amino acid network and its scoring application in protein-protein docking.

Protein-protein complex, composed of hydrophobic and hydrophilic residues, can be divided into hydrophobic and hydrophilic amino acid network structures respectively. In this paper, we are interested in analyzing these two different types of networks and find that these networks are of small-world properties. Due to the characteristic complementarity of the complex interfaces, protein-protein docking can be viewed as a particular network rewiring. These networks of correct docked complex conformations have much more increase of the degree values and decay of the clustering coefficients than those of the incorrect ones. Therefore, two scoring terms based on the network parameters are proposed, in which the geometric complementarity, hydrophobic-hydrophobic and polar-polar interactions are taken into account. Compared with a two-term energy function, a simple scoring function HPNet which includes the two network-based scoring terms shows advantages in two aspects, not relying on energy considerations and better discrimination. Furthermore, combing the network-based scoring terms with some other energy terms, a new multi-term scoring function HPNet-combine can also make some improvements to the scoring function of RosettaDock.

A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads.

AIM: To develop a novel high-throughput format assay to monitor the integrase (IN) strand transfer (ST) reaction in vitro and apply it to a reaction character study and the identification of antiviral drugs. METHODS: The donor DNA duplex, with a sequence identical to the U5 end of HIV-1 long terminal repeats, is labeled at its 5' end with biotin (BIO). The target DNA duplex is labeled at its 3' end with digoxin (DIG). IN mediates the integration of donor DNA into target DNA and results in a 5' BIO and 3' DIG-labeled duplex DNA product. Streptavidin-coated magnetic beads were used to capture the product, and the amount of DIG was measured as the ST reaction product. The assay was optimized in 96-well microplate format for high-throughput screening purpose. Moreover, the assay was applied in a ST reaction character study, and the efficiency of the assay in the identification of antiviral compounds was tested. RESULTS: The end-point values, measured as absorbance at 405 nm was approximately 1.5 for the IN-mediated ST reaction as compared with no more than 0.05 of background readings. The ST reaction character and the half maximal inhibitory concentration (IC50) values of 2 known IN inhibitors obtained in our assay were similar to previously reported results using other assays. The evaluation parameter Z' factor for this assay ranged from 0.6 to 0.9. CONCLUSION: The assay presented here has been proven to be rapid, sensitive, and specific for the detection of IN ST activity, the reaction character study, as well as for the identification of antiviral drugs targeting IN.

Prediction of PK-specific phosphorylation site based on information entropy.

Phosphorylation is a crucial way to control the activity of proteins in many eukaryotic organisms in vivo. Experimental methods to determine phosphorylation sites in substrates are usually restricted by the in vitro condition of enzymes and very intensive in time and labor. Although some in silico methods and web servers have been introduced for automatic detection of phosphorylation sites, sophisticated methods are still in urgent demand to further improve prediction performances. Protein primary sequences can help predict phosphorylation sites catalyzed by different protein kinase and most computational approaches use a short local peptide to make prediction. However, the useful information may be lost if only the conservative residues that are not close to the phosphorylation site are considered in prediction, which would hamper the prediction results. A novel prediction method named IEPP (Information-Entropy based Phosphorylation Prediction) is presented in this paper for automatic detection of potential phosphorylation sites. In prediction, the sites around the phosphorylation sites are selected or excluded by their entropy values. The algorithm was compared with other methods such as GSP and PPSP on the ABL, MAPK and PKA PK families. The superior prediction accuracies were obtained in various measurements such as sensitivity (Sn) and specificity (Sp). Furthermore, compared with some online prediction web servers on the new discovered phosphorylation sites, IEPP also yielded the best performance. IEPP is another useful computational resource for identification of PK-specific phosphorylation sites and it also has the advantages of simpleness, efficiency and convenience.

Construction and application of the weighted amino acid network based on energy.

A method is proposed to construct the weighted amino acid network. The weight of the link is based on the contact energy between residues. For the 197 proteins with low homology, the "small-world" property was studied based on this method. Additionally, analyses were carried out for the statistic characteristics of the network parameters, the influence of the weight on the network parameters, the network parameter difference of amino acids, and the links between the hydrophobic and hydrophilic residues. Using this method, we studied the network parameter change for the protein chymotrypsin inhibitor 2 (CI2) on its high-temperature unfolding pathway. It is found that the unfolding of the protein is mainly exhibited as the derogation of the hydrophobic core and the shortest path length rise in the unfolding process. This work is helpful for studies of protein folding and the relationship between structure and function using complex network theory.

High-throughput real-time assay based on molecular beacons for HIV-1 integrase 3'-processing reaction.

AIM: To develop a high-throughput real-time assay based on molecular beacons to monitor the integrase 3'-processing reaction in vitro and apply it to inhibitor screening. METHODS: The recombinant human immunodeficiency virus (HIV)-1 integrase (IN) is incubated with a 38 mer oligonucleotide substrate, a sequence identical to the U5 end of HIV-1 long terminal repeats (LTR). Based on the fluorescence properties of molecular beacons, the substrate is designed to form a stem-loop structure labeled with a fluorophore at the 5' end and a quencher at the 3' end. IN cleaves the terminal 3'-dinucleotide containing the quencher, resulting in an increase in fluorescence which can be monitored on a spectrofluorometer. To optimize this assay, tests were performed to investigate the effects of substrates, enzyme and the metal ion concentrations on the IN activity and optimal parameters were obtained. Moreover, 2 IN inhibitors were employed to test the performance of this assay in antiviral compound screening. RESULTS: The fluorescent intensity of the reaction mixture varies linearly with time and is proportional to the velocity of the 3'-processing reaction. Tests were performed and the results showed that the optimal rate was obtained for a reaction mixture containing 50 mg/L recombinant HIV-1 IN, 400 nmol/L substrate, and 10 mmol/L Mn(2+). The IN 3'-processing reaction under the optimal conditions showed a more than 18-fold increase in the fluorescence intensity compared to the enzyme-free control. The IC50 values of the IN inhibitors obtained in our assay were similar to the values obtained from a radiolabeled substrate assay. CONCLUSION: Our results demonstrated that this is a fast, reliable, and sensitive method to monitor HIV IN 3'-processing reaction and that it can be used for inhibitor screening.

Protein design based on the relative entropy.

An approach to protein design is proposed based on the relative entropy and a reduced amino acid alphabet. In this approach, the relative entropy is used as a minimization object function. The method has been tested on a real protein's off-lattice model successfully, and the results are similar to those obtained from other design studies. It can be applied as a uniform frame for both folding and inverse folding of protein. An iterative calculation method of the ensemble average of the contact strength is proposed at the same time.

Differential interaction of cardiac, skeletal muscle, and yeast tropomyosins with fluorescent (pyrene235) yeast actin.

To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase, suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, whereas that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca(+2)-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin +/- troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform-specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.

Constructing HIV-1 integrase tetramer and exploring influences of metal ions on forming integrase-DNA complex.

HIV-1 integrase (IN) is essential for the replication of HIV-1 in human cells. At present, the complete structure of complex IN-DNA has not been resolved. In this paper, a HIV-1 IN tetramer model was built with homology modeling and molecular dynamics simulation approach, in which two Mg2+ ions were reasonably located in each catalytic core domain. Moreover, it was found that the AB and CD chains of HIV-1 IN tetramer were different in the structures and metal ions of HIV-1 IN tetramer might have great influences on DNA locating on IN. These findings may provide a more complete structural basis for guiding drug discovery and revealing integration mechanism.

Exploring binding mode for styrylquinoline HIV-1 integrase inhibitors using comparative molecular field analysis and docking studies.

AIM: To understand pharmacophore properties of styrylquinoline derivatives and to design inhibitors of HIV-1 integrase. METHODS: Comparative molecular field analysis (CoMFA) was performed to analyze three-dimensional quantitative structure-activity relationship (3D-QSAR) of styrylquinoline derivatives. Thirty-eight compounds were randomly divided into a training set of 28 compounds and a test set of 10 compounds. The stability of 3D-QSAR models was proved by the analysis of cross-validated and non-cross-validated methods. Moreover, the binding mode of these compounds and integrase was constructed by AutoDock program. RESULTS: The CoMFA model of the training compounds was reasonably predicted with cross-validated coefficient (q2) and conventional (r2) values (up to 0.696 and 0.754). Then the model was validated by the test set. The resulting CoMFA maps visualized structural requirements for the biological activity of these inhibitors. Docking results showed that a carboxyl group at C-7 and a hydroxyl group at C-8 in the quinoline subunit, bound closely to the divalent metal cofactor (Mg2+) around the integrase catalytic site. Moreover, there is a linear correlation between the binding energy of the inhibitors with integrase and their inhibitory effect. CONCLUSIONS: The present study indicated that the CoMFA model together with docking results could give us helpful hints for drug design as well as interpretation of the binding affinity between these inhibitors and integrase.