Entropy Hotspots for the Binding of Intrinsically Disordered Ligands to a Receptor Domain.

Proline-rich motifs (PRMs) are widely used for mediating protein-protein interactions with weak binding affinities. Because they are intrinsically disordered when unbound, conformational entropy plays a significant role for the binding. However, residue-level differences of the entropic contribution in the binding of different ligands remain not well understood. We use all-atom molecular dynamics simulation and the maximal information spanning tree formalism to analyze conformational entropy associated with the binding of two PRMs, one from the Abl kinase and the other from the nonstructural protein 1 of the 1918 Spanish influenza A virus, to the N-terminal SH3 (nSH3) domain of the CrkII protein. Side chains of the stably folded nSH3 experience more entropy change upon ligand binding than the backbone, whereas PRMs involve comparable but heterogeneous entropy changes among the backbone and side chains. In nSH3, two conserved nonpolar residues forming contacts with the PRM experience the largest side-chain entropy loss. In contrast, the C-terminal charged residues of PRMs that form polar contacts with nSH3 experience the greatest side-chain entropy loss, although their "fuzzy" nature is attributable to the backbone that remains relatively flexible. Thus, residues that form high-occupancy contacts between nSH3 and PRM do not reciprocally contribute to entropy loss. Furthermore, certain surface residues of nSH3 distal to the interface with PRMs gain entropy, indicating a nonlocal effect of ligand binding. Comparing between the PRMs from cAbl and nonstructural protein 1, the latter involves a larger side-chain entropy loss and forms more contacts with nSH3. Consistent with experiments, this indicates stronger binding of the viral ligand at the expense of losing the flexibility of side chains, whereas the backbone experiences less entropy loss. The entropy "hotspots" as identified in this study will be important for tuning the binding affinity of various ligands to a receptor.

Molecular Basis of the Ternary Interaction between NS1 of the 1918 Influenza A Virus, PI3K, and CRK.

The 1918 influenza A virus (IAV) caused the worst flu pandemic in human history. Non-structural protein 1 (NS1) is an important virulence factor of the 1918 IAV and antagonizes host antiviral immune responses. NS1 increases virulence by activating phosphoinositide 3-kinase (PI3K) via binding to the p85ß subunit of PI3K. Intriguingly, unlike the NS1 of other human IAV strains, 1918 NS1 hijacks another host protein, CRK, to form a ternary complex with p85ß, resulting in hyperactivation of PI3K. However, the molecular basis of the ternary interaction between 1918 NS1, CRK, and PI3K remains elusive. Here, we report the structural and thermodynamic bases of the ternary interaction. We find that the C-terminal tail (CTT) of 1918 NS1 remains highly flexible in the complex with p85ß. Thus, the CTT of 1918 NS1 in the complex with PI3K can efficiently hijack CRK. Notably, our study indicates that 1918 NS1 enhances its affinity to p85ß in the presence of CRK, which might result in enhanced activation of PI3K. Our results provide structural insight into how 1918 NS1 hijacks two host proteins simultaneously.

Molecular recognition of a host protein by NS1 of pandemic and seasonal influenza A viruses.

The 1918 influenza A virus (IAV) caused the most severe flu pandemic in recorded human history. Nonstructural protein 1 (NS1) is an important virulence factor of the 1918 IAV. NS1 antagonizes host defense mechanisms through interactions with multiple host factors. One pathway by which NS1 increases virulence is through the activation of phosphoinositide 3-kinase (PI3K) by binding to its p85ß subunit. Here we present the mechanism underlying the molecular recognition of the p85ß subunit by 1918 NS1. Using X-ray crystallography, we determine the structure of 1918 NS1 complexed with p85ß of human PI3K. We find that the 1918 NS1 effector domain (1918 NS1ED) undergoes a conformational change to bind p85ß. Using NMR relaxation dispersion and molecular dynamics simulation, we identify that free 1918 NS1ED exists in a dynamic equilibrium between p85ß-binding-competent and -incompetent conformations in the submillisecond timescale. Moreover, we discover that NS1ED proteins of 1918 (H1N1) and Udorn (H3N2) strains exhibit drastically different conformational dynamics and binding kinetics to p85ß. These results provide evidence of strain-dependent conformational dynamics of NS1. Using kinetic modeling based on the experimental data, we demonstrate that 1918 NS1ED can result in the faster hijacking of p85ß compared to Ud NS1ED, although the former has a lower affinity to p85ß than the latter. Our results suggest that the difference in binding kinetics may impact the competition with cellular antiviral responses for the activation of PI3K. We anticipate that our findings will increase the understanding of the strain-dependent behaviors of influenza NS1 proteins.

The structure and conformational plasticity of the nonstructural protein 1 of the 1918 influenza A virus.

Nonstructural protein 1 (NS1) is a multifunctional virulence factor of influenza virus. The effector domain (ED) of influenza viruses is capable of binding to a variety of host factors, however, the molecular basis of the interactions remains to be investigated. The isolated NS1-ED exists in equilibrium between the monomer and homodimer. Although the structural diversity of the dimer interface has been well-characterized, limited information is available regarding the internal conformational heterogeneity of the monomeric NS1-ED. Here, we present the solution NMR structure of the NS1-ED W187R of the 1918 influenza A virus, which caused the "Spanish flu." Structural plasticity is an essential property to understand the molecular mechanism by which NS1-ED interacts with multiple host proteins. Structural comparison with the NS1-ED from influenza A/Udorn/1972 (Ud) strain revealed a similar overall structure but a distinct conformational variation and flexibility. Our results suggest that conformational flexibility of the NS1-ED might differ depending on the influenza strain.

3C-SiC Nanowires In-Situ Modified Carbon/Carbon Composites and Their Effect on Mechanical and Thermal Properties.

An in-situ, catalyst-free method for synthesizing 3C-SiC ceramic nanowires (SiCNWs) inside carbon⁻carbon (C/C) composites was successfully achieved. Obtained samples in different stages were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and Raman scattering spectroscopy. Results demonstrated that the combination of sol-gel impregnation and carbothermal reduction was an efficient method for in-situ SiCNW synthesis, inside C/C composites. Thermal properties and mechanical behaviors-including out-of-plane and in-plane compressive strengths, as well as interlaminar shear strength (ILLS) of SiCNW modified C/C composites-were investigated. By introducing SiCNWs, the initial oxidation temperature of C/C was increased remarkably. Meanwhile, out-of-plane and in-plane compressive strengths, as well as interlaminar shear strength (ILLS) of C/C composites were increased by 249.3%, 109.2%, and 190.0%, respectively. This significant improvement resulted from simultaneous reinforcement between the fiber/matrix (F/M) and matrix/matrix (M/M) interfaces, based on analysis of the fracture mechanism.

Structure-guided design of a potent peptide inhibitor targeting the interaction between CRK and ABL kinase.

CT-10 regulator of kinase (CRK) proteins play important roles in human cancer metastasis and invasion. Moreover, CRK proteins are the major phosphorylation substrates of ABL kinase and its oncogenic mutant BCR-ABL kinase. The interaction between CRK and BCR-ABL plays important roles in chronic myeloid leukemia. Hence, inhibiting the interaction of CRK with BCR-ABL is an attractive way to attenuate cancer metastasis. Herein, we report the development of a peptide inhibitor, PRM-3, targeting the interaction between CRK-II and ABL kinase. PRM-3 binds to the N-terminal SH3 (nSH3) domain in CRK-II with a 10 nM affinity and prevents the interaction between CRK-II and ABL kinase. An in vitro biochemical assay demonstrated that PRM-3 inhibits the ABL-dependent phosphorylation of CRK-II more effectively than imatinib. Remarkably, PRM-3 also inhibited the CRK phosphorylation by T315I-ABL kinase, which is resistant to all first- and second-generation tyrosine kinase inhibitors. Our study provides a promising alternative approach to overcome the drug resistance of ABL kinase.

Molecular Mechanisms of Tight Binding through Fuzzy Interactions.

Many intrinsically disordered proteins (IDPs) form fuzzy complexes upon binding to their targets. Although many IDPs are weakly bound in fuzzy complexes, some IDPs form high-affinity complexes. One example is the nonstructural protein 1 (NS1) of the 1918 Spanish influenza A virus, which hijacks cellular CRKII through the strong binding affinity (Kd ∼10 nM) of its proline-rich motif (PRMNS1) to the N-terminal Src-homology 3 domain of CRKII. However, its molecular mechanism remains elusive. Here, we examine the interplay between structural disorder of a bound PRMNS1 and its long-range electrostatic interactions. Using x-ray crystallography and NMR spectroscopy, we found that PRMNS1 retains substantial conformational flexibility in the bound state. Moreover, molecular dynamics simulations showed that structural disorder of the bound PRMNS1 increases the number of electrostatic interactions and decreases the mean distances between the positively charged residues in PRMNS1 and the acidic residues in the N-terminal Src-homology 3 domain. These results are analyzed using a polyelectrostatic model. Our results provide an insight into the molecular recognition mechanism for a high-affinity fuzzy complex.

The Molecular Mechanisms Underlying the Hijack of Host Proteins by the 1918 Spanish Influenza Virus.

The 1918 Spanish influenza A virus (IAV) caused one of the most serious pandemics in history. The nonstructural protein 1 (NS1) of the 1918 IAV hijacks the interaction between human CrkII and JNK1. Little is, however, known about its molecular mechanism. Here, we performed X-ray crystallography, NMR relaxation dispersion experiment, and fluorescence spectroscopy to determine the structural, kinetic, and thermodynamic mechanisms underlying the hijacking of CrkII by 1918 IAV NS1. We observed that the interaction between a proline-rich motif in NS1 and the N-terminal SH3 domain of CrkII displays strikingly rapid kinetics and exceptionally high affinity with 100-fold faster kon and 3300-fold lower Kd compared to those for the CrkII-JNK1 interaction. These results provide molecular insight into the mechanism by which 1918 IAV NS1 hijacks CrkII and disrupts its interactions with critical cellular signaling proteins.

Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

Kinetic Insights into the Binding between the nSH3 Domain of CrkII and Proline-Rich Motifs in cAbl.

The interaction between CrkII and cAbl is implicated in diverse cellular processes. This interaction starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to the proline-rich motifs of cAbl (PRMscAbl). Despite its critical importance, the detailed binding mechanism between the nSH3 domain and PRMs remains elusive. In this study, we used nuclear magnetic resonance Carr-Purcell-Meiboom-Gill relaxation dispersion experiment to study the binding kinetics between the nSH3 domain of CrkII and PRMscAbl. Our results highlight that the nSH3 domain binds to three PRMscAbl with very high on- and off-rate constants, indicating the transient nature of the binding. To further characterize the binding transition state, we conducted the Eyring and linear free energy relationship analyses using temperature-dependent kinetic data. These data indicate that the binding transition state of the nSH3 domain and PRM is accompanied by small activation enthalpy, owing to partial desolvation of the transition state. These results also highlight the similarity between the transition and free states, in terms of structure and energetics. Although the binding of the nSH3 domain and PRM displays the features consistent with a diffusion-limited process within our experimental conditions, further tests are necessary to determine if the binding is a true diffusion-limited process.

Analysis of the interferences in quantitation of a site-specifically PEGylated exendin-4 analog by the Bradford method.

Protein modification has been found to affect the estimation of protein concentration in some of the traditional dye-based absorbance measurements. In this work, a distinct reduction in A595 was observed during the quantitation of a PEGylated exendin-4 analogue (Ex4C) by the Bradford method and the PEGylation process was found to interfere with the measurement. Lys(12), Arg(20), and Lys(27) were further proved to be the major amino acids that functioned as dye-binding sites. The shielding effect produced by the large polymer was demonstrated to depend on the length of PEG that was used for modification.

[Percutaneous release of trigger finger with L shaped hollow needle knife].

OBJECTIVE: To investigate the effectiveness of a percutaneous release with L shaped hollow needle knife in treating trigger finger. METHODS: Between September 2007 and September 2009, 160 patients with trigger fingers (202 fingers) were treated by percutaneous release with L shaped hollow needle knife. There were 47 males and 113 females with a mean age of 55 years (range, 12-68 years). The disease duration was 2 weeks to 1 year. Affected fingers included 58 thumbs, 20 index fingers, 46 middle fingers, 60 ring fingers, and 18 little fingers. According to Quinnell grading, 63 fingers were classified as grade III, 126 fingers as grade IV, and 13 fingers as grade V. A1 pulley was released during operation and steroid was injected after release procedure using the same needle. RESULTS: The mean operation time was 8.2 minutes (range, 5-19 minutes), and no complication occurred. All the patients were followed up 1 year to 3 years and 6 months (mean, 1.6 years). The patients still felt pain in 36 fingers at 1 week after operation, which were relieved after oral administration of non-steroidal anti-inflammatory drug. Twenty-five fingers had snapping or locking in flexion-extension motion; 5 fingers recovered at 1 month after operation and 20 fingers had no obvious improvement; of 20 fingers, symptom was alleviated in 10 fingers, and was not alleviated in 10 fingers after re-release with L shaped hollow needle knife. According to Quinnell grading for efficacy evaluation at 6 months after operation, the results were excellent in 165 fingers, good in 27 fingers, poor in 10 fingers with an excellent and good rate of 95.0%. CONCLUSION: The percutaneous release with L shaped hollow needle knife is a safe and effective procedure in treating trigger finger with low complications.

[Efficacy of water knife needle release combined with bone peptide injection for heel pain].

OBJECTIVE: To evaluate the efficacy of water knife needle release combined with bone peptide injection in the management of heel pain. METHODS: Thirty-five patients with unilateral heel pain were treated with water knife needle release and bone peptide injection under local anesthesia. The deep tissue with the tenderness was released in the operation, and the result was evaluated 1 week after the surgery to decide whether to conduct another surgery. No more than 3 treatment sessions were administered. The efficacy was evaluated according to nimodipine method by the principles of Chinese clinical drug guidance, and the complications of the surgery were observed. RESULTS: Six months after the surgery, 28 cases had excellent results, 3 had good outcomes, 2 showed improvement, and 2 failed to respond favorably, with a rate of good and excellent result of 94.2%. No adverse side effect was recorded in the follow up of the patients. CONCLUSION: Water needle knife release combined with bone peptide injection can produce a good result in the treatment of heel pain.