Multistep kinetics of the U1A-SL2 RNA complex dissociation

The U1A-SL2 RNA complex is a model system for studying interactions between RNA and the RNA recognition motif (RRM), which is one of the most common RNA binding domains. We report here kinetic studies of dissociation of the U1A-SL2 RNA complex, using laser temperature jump and stopped-flow fluorescence methods with U1A proteins labeled with the intrinsic chromophore tryptophan. An analysis of the kinetic data suggests three phases of dissociation with time scales of ∼ 100 μs, ∼ 50 ms, and ∼ 2 s. We propose that the first step of dissociation is a fast rearrangement of the complex to form a loosely bound complex. The intermediate step is assigned to be the dissociation of the U1A-SL2 RNA complex, and the final step is assigned to a reorganization of the U1A protein structure into the conformation of the free protein. These assignments are consistent with previous proposals based on thermodynamic, NMR, and surface plasmon resonance experiments and molecular dynamics simulations. Together, these results begin to build a comprehensive model of the complex dynamic processes involved in the formation and dissociation of an RRM-RNA complex.     

Dynamic control of allosteric antagonism of leukocyte function antigen-1 and intercellular adhesion molecule-1 interaction

Leukocyte function associated antigen-1 (LFA-1) plays a critical role in T cell migration and has been recognized as a therapeutic target for immune disorders. Several classes of small molecule antagonists have been developed to block LFA-1 interaction with intercellular adhesion molecule-1 (ICAM-1). Recent structural studies show that the antagonists bind to an allosteric site in the I-domain of LFA-1. However, it is not yet clear how these small molecules work as antagonists since no significant conformational change is observed in the I-domain-antagonist complex structures. Here we present a computational study suggesting how these allosteric antagonists affect the dynamics of the I-domain. The lowest frequency vibrational mode calculated from an LFA-1 I-domain structure shows large scale "coil-down" motion of the C-terminal alpha7 helix, which may lead to the open form of the I-domain. The presence of an allosteric antagonist greatly reduces this motion of the alpha7 helix as well as other parts of the I-domain. Thus, our study suggests that allosteric antagonists work by eliminating breathing motion that leads to the open conformation of the I-domain.     

Molecular dynamics-solvated interaction energy studies of protein-protein interactions: the MP1-p14 scaffolding complex

Using the MP1-p14 scaffolding complex from the mitogen-activated protein kinase signaling pathway as model system, we explored a structure-based computational protocol to probe and characterize binding affinity hot spots at protein-protein interfaces. Hot spots are located by virtual alanine-scanning consensus predictions over three different energy functions and two different single-structure representations of the complex. Refined binding affinity predictions for select hot-spot mutations are carried out by applying first-principle methods such as the molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) to the molecular dynamics (MD) trajectories for mutated and wild-type complexes. Here, predicted hot-spot residues were actually mutated to alanine, and crystal structures of the mutated complexes were determined. Two mutated MP1-p14 complexes were investigated, the p14(Y56A)-mutated complex and the MP1(L63A,L65A)-mutated complex. Alternative ways to generate MD ensembles for mutant complexes, not relying on crystal structures for mutated complexes, were also investigated. The SIE function, fitted on protein-ligand binding affinities, gave absolute binding affinity predictions in excellent agreement with experiment and outperformed standard MM-GBSA predictions when tested on the MD ensembles of Ras-Raf and Ras-RalGDS protein-protein complexes. For wild-type and mutant MP1-p14 complexes, SIE predictions of relative binding affinities were supported by a yeast two-hybrid assay that provided semiquantitative relative interaction strengths. Results on the MP1-mutated complex suggested that SIE predictions deteriorate if mutant MD ensembles are approximated by just mutating the wild-type MD trajectory. The SIE data on the p14-mutated complex indicated feasibility for generating mutant MD ensembles from mutated wild-type crystal structure, despite local structural differences observed upon mutation. For energetic considerations, this would circumvent costly needs to produce and crystallize mutated complexes. The sensitized protein-protein interface afforded by the p14(Y56A) mutation identified here has practical applications in screening-based discovery of first-generation small-molecule hits for further development into specific modulators of the mitogen-activated protein kinase signaling pathway.     

A study of collective atomic fluctuations and cooperativity in the U1A-RNA complex based on molecular dynamics simulations

Cooperative interactions play an important role in recognition and binding in macromolecular systems. In this study, we find that cross-correlated atomic fluctuations can be used to identify cooperative networks in a protein–RNA system. The dynamics of the RRM-containing protein U1A–stem loop 2 RNA complex have been calculated theoretically from a 10 ns molecular dynamics (MD) simulation. The simulation was analyzed by calculating the covariance matrix of all atomic fluctuations. These matrix elements are then presented in the form of a two-dimensional grid, which displays fluctuations on a per residue basis. The results indicate the presence of strong, selective cross-correlated fluctuations throughout the RRM in U1A–RNA. The atomic fluctuations correspond well with previous biophysical studies in which a multiplicity of cooperative networks have been reported and indicate that the various networks identified in separate individual experiments are fluctuationally correlated into a hyper-network encompassing most of the RRM. The calculated results also correspond well with independent results from a statistical covariance analysis of 330 aligned RRM sequences. This method has significant implications as a predictive tool regarding cooperativity in the protein–nucleic acid recognition process.     

Effect of Interdomain Linker Length on an Antagonistic Folding-Unfolding Equilibrium between Two Protein Domains

Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (ΔG_X). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. ΔG_X values are determined by measuring the extent to which Co²⁺ binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that ΔG_X increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.     

Insight into the key active sites of afChiA1 based on molecular dynamics simulations and free energy calculations

In this study, the binding of the enzyme chitinase A1 (afChiA1) from the plant-type Aspergillus fumigatus with four potent inhibitors, allosamidin (ASM), acetazolamide (AZM), 8-chloro-theophylline (CTP) and kinetin (KIT) is investigated by molecular docking, molecular dynamics simulation and binding free energy calculation. The results reveal that the electrostatic interactions play an important role in the stabilisation of the binding of afChiA1 with inhibitors. Based on the binding energy of afChiA1-ligands, the key residues (Gln37 and Trp312) in the active binding pocket of the complex systems are confirmed by molecular mechanics/Poisson–Boltzmann surface area method, and the active inhibitors, ASM and AZM, both could form strong interaction with Gln37 and Trp312, and the non-active ligands, CTP and KIT, could not interact with these two residues, which is consistent with the result of experimental report. Then, it is identified that Gln37 and Trp312 should be one of the important active site residues of afChiA1.     

Sequence context dependence of tandem guanine:adenine mismatch conformations in RNA: a continuum solvent analysis

Guanine:adenine (G:A) mismatches and in particular tandem G:A (tG:A) mismatches are frequently observed in biological RNA molecules and can serve as sites for tertiary interaction, metal binding and protein recognition. Depending on the surrounding sequence tG:A mismatches can adopt different basepairing topologies. In the sequence context (5'-) GGAC (tandem G:A in bold) a face-to-face (imino or Watson-Crick-like) pairing is preferred whereas in the CGAG context, G and A adopt a sheared arrangement. Systematic conformational searches with a generalized Born continuum model and molecular dynamics simulations including explicit water molecules and ions have been used to generate face-to-face and sheared tG:A mismatches in both CGAG and GGAC sequence contexts. Conformations from both approaches were evaluated using the same force field and a Poisson-Boltzmann continuum solvent model. Although the substate analysis predicted the sheared arrangement to be energetically preferred in both sequence contexts, a significantly greater preference of the sheared form was found for the CGAG context. In agreement with the experimental observation, the analysis of molecular dynamics trajectories indicated a preference of the sheared form in the case of the CGAG-context and a favorization of the face-to-face form in the case of the GGAC context. The computational studies allowed to identify energetic contributions that stabilize or destabilize the face-to-face and sheared tandem mismatch topologies. The calculated nonpolar solvation and Lennard-Jones packing interaction were found to stabilize the sheared topology independent of the sequence context. Electrostatic contributions are predicted to make the most significant contribution to the sequence context dependence on the structural preference of tG:A mismatches.     

Characterization of domain-peptide interaction interface: a case study on the amphiphysin-1 SH3 domain

Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was used to assess the performances of different kernel functions in SVM and different combinations of the MIECs. The best SVM classifier gave satisfactory predictions for the test set, indicated by average prediction accuracy rates of 78% and 91% for the binding and non-binding peptides, respectively. We also showed that the performance of our approach on both binding affinity prediction and binder/non-binder classification was superior to the performances of the conventional MM/Poisson-Boltzmann solvent-accessible surface area and MM/generalized Born solvent-accessible surface area calculations. Our study demonstrates that the analysis of the MIECs between peptides and the SH3 domain can successfully characterize the binding interface, and it provides a framework to derive integrated prediction models for different domain-peptide systems.     

Molecular dynamics simulations and free energy analyses on the dimer formation of an amyloidogenic heptapeptide from human beta2-microglobulin: implication for the protofibril structure

Amyloid formation is associated with many neurodegenerative diseases. Recent findings suggest that early oligomeric aggregates could be major sources of toxicity. We present a computational investigation of the first step of amyloid initiation-dimer formation of a seven residue peptide (NHVTLSQ) from human beta2-microglobulin at pH 2.0, which renders +2.0 units charges to each peptide. A total of over 1.2 micros of simulations with explicit solvent and 1.0 micros of simulations with implicit solvent were conducted. Main-chain conformational restraint was applied to facilitate the formation of ordered dimers. An antiparallel beta-sheet with six main-chain hydrogen bonds was dominant in the implicit solvent simulations. In contrast, no stable dimers were observed in the two negative controls, the mouse heptapeptide (KHDSMAE, +3.0 units charges) and the scrambled human heptapeptide (QVLHTSN). Explicit solvent simulations presented a more complex scenario. The wild-type human heptapeptide formed predominantly antiparallel beta-sheets ( approximately 38%) although parallel ones ( approximately 12%) were also observed. Hydrophobic contacts preceded hydrogen bond saturation in the majority of the association events in the explicit solvent simulations, highlighting the important role of hydrophobic interaction in amyloid initiation. The fact that the mouse dimer dissociated immediately after the removal of conformational restraint suggests that the higher conformational entropy barrier, along with the stronger charge repulsion and weaker hydrophobic interaction, contributed to its inability to form amyloid fibril. The closeness of positive charge pairs in the dimers of the scrambled human heptapeptide may prohibit further beta-sheet extension and fibril growth. Combining the results from simulations and free energy analyses, we propose that the building block for this amyloid fibril is an antiparallel dimer with a two-residue register shift and six main-chain hydrogen bonds. A double-layer protofibril structure is also proposed in which two antiparallel beta-sheets face each other and are held together by hydrophobic staples and hydrogen bonds of the polar side-chains.     

X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases

Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9 kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1 kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants.     

RRM proteins interacting with the cap region of topoisomerase I

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).