•Promiscuous and specific Eph-ephrin binding between cells controls communication.•Studied conformational ensembles and energies of sixteen Eph - ephrins complexes.•The process of recognition involve progressive conformational selection events.•Dynamics control specificity, anchoring residues may offer promiscuity.•Conformational dynamics may simultaneously allow promiscuous and specific binding.Eph–ephrin interactions control the signal transduction between cells and play an important role in carcinogenesis and other diseases. The interactions between Eph receptors and ephrins of the same subclass are promiscuous; there are cross-interactions between some subclasses, but not all. To understand how Eph–ephrin interactions can be both promiscuous and specific, we investigated sixteen energy landscapes of four Eph receptors (A2, A4, B2, and B4) interacting with four ephrin ligands (A1, A2, A5, and B2). We generated conformational ensembles and recognition energy landscapes starting from separated Eph and ephrin molecules and proceeding up to the formation of Eph–ephrin complexes. Analysis of the Eph–ephrin recognition trajectories and the co-evolution entropy of 400 ligand binding domains of Eph receptor and 241 ephrin ligands identified conserved residues during the recognition process. Our study correctly predicted the promiscuity and specificity of the interactions and provided insights into their recognition. The dynamic conformational changes during Eph–ephrin recognition can be described by progressive conformational selection and population shift events, with two dynamic salt bridges between EphB4 and ephrin-B2 contributing to the specific recognition. EphA3 cancer-related mutations lowered the binding energies. The specificity is not only controlled by the final stage of the interaction across the protein–protein interface, but also has large contributions from binding kinetics with the help of dynamic intermediates along the pathway from the separated Eph and ephrin to the Eph–ephrin complex.

To better understand the structural origins of inhibitor selectivity of human phosphodieasterase families (PDEs 1–11), here we report the X-ray crystal structure of PDE2 in complex with a highly selective, nanomolar inhibitor (BAY60-7550) at 1.9 Å resolution, and the structure of apo PDE2 at 2.0 Å resolution. The crystal structures reveal that the inhibitor binds to the PDE2 active site by using not only the conserved glutamine-switch mechanism for substrate binding, but also a binding-induced, hydrophobic pocket that was not reported previously. In silico affinity profiling by molecular docking indicates that the inhibitor binding to this pocket contributes significantly to the binding affinity and thereby improves the inhibitor selectivity for PDE2. Our results highlight a structure-based design strategy that exploits the potential binding-induced pockets to achieve higher selectivity in the PDE inhibitor development.

Protein folding, stability, and function are usually influenced by pH. And free energy plays a fundamental role in analysis of such pH-dependent properties. Electrostatics-based theoretical framework using dielectric solvent continuum model and solving Poisson-Boltzmann equation numerically has been shown to be very successful in understanding the pH-dependent properties. However, in this approach the exact computation of pH-dependent free energy becomes impractical for proteins possessing more than several tens of ionizable sites (e.g. > 30), because exact evaluation of the partition function requires a summation over a vast number of possible protonation microstates. Here we present a method which computes the free energy using the average energy and the protonation probabilities of ionizable sites obtained by the well-established Monte Carlo sampling procedure. The key feature is to calculate the entropy by using the protonation probabilities. We used this method to examine a well-studied protein (lysozyme) and produced results which agree very well with the exact calculations. Applications to the optimum pH of maximal stability of proteins and protein-DNA interactions have also resulted in good agreement with experimental data. These examples recommend our method for application to the elucidation of the pH-dependent properties of proteins.

Pre-transfer editing pathway in Valyl-tRNA synthetase (ValRS) is a very important process to maintain the high fidelity of protein synthesis. However, molecular basis for this pathway remains unclear. Here we employed molecular dynamics (MD) simulation to study two complexes, ValRS·tRNAval·Val-AMP (complex V) and ValRS·tRNAval·Thr-AMP (complex T), and compared their simulation trajectories, in order to understand how the pre-transfer editing pathway is triggered by the noncognate substrate Thr-AMP. The MD simulations showed that the binding of Thr-AMP to ValRS led to different motions from those in complex V: clockwise rotation of the editing domain along the hinge region, and strong motions in the catalytic domain, especially in KMSKS loop. We found that the changed motion of Trp495 induced by Thr-AMP serves as a signal to discriminate Thr-AMP from Val-AMP, and the rigid 491ILFL494 segment then propagates this signal from Trp495 to Asp490 and induces dissociation of the salt-bridge Asp490-Arg346 and formation of the salt-bridge Glu189-Lys533. The change in salt-bridges alters the motion of KMSKS loop and the editing domain, and eventually triggers the pre-transfer editing pathway. This study provides a model for the molecular trigger of the pre-transfer editing pathway in ValRS, and is useful for further exploring this process.

ATB107 is a potent inhibitor of indole-3-glycerol phosphate synthase (IGPS). It can effectively inhibit the growth of clinical isolates of drug-resistant Mycobacterium tuberculosis strains as well as M. tuberculosis H37Rv. To investigate the mechanism of ATB107 action in M. tuberculosis, two-dimensional gel electrophoresis coupled with MALDI-TOF-MS analysis (2-DE-MS) was performed to illustrate alterations in the protein expression profile in response to ATB107. Results show that ATB107 affected tryptophan biosynthesis by decreasing the expression of protein encoded by Rv3246c, the transcriptional regulatory protein of MtrA belonging to the MtrA-MtrB two-component regulatory system, in both drug-sensitive and drug-resistant virulent strains. ATB107 might present a stress condition similar to isoniazid (INH) or ethionamide for M. tuberculosis since the altered expression in response to ATB107 of some genes, such as Rv3140, Rv2243, and Rv2428, is consistent with INH or ethionamide treatment. After incubation with ATB107, the expression of 2 proteins encoded by Rv0685 and Rv2624c was down-regulated while that of protein encoded by Rv3140 was up-regulated in all M. tuberculosis strains used in this study. This may be the common response to tryptophan absence; however, relations to ATB107 are unknown and further evaluation is warranted.

Indole-3-glycerol phosphate synthase (IGPS) is a representative of (β/α)8-barrel proteins—the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (βα)8-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (β/α)8-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (β/α)8-barrel scaffold.