Melodia: A Python Library for Protein Structure Analysis.

SUMMARY: Analysing protein structure similarities is an important step in protein engineering and drug discovery. Methodologies that are more advanced than simple RMSD are available but often require extensive mathematical or computational knowledge for implementation. Grouping and optimising such tools in an efficient open-source library increases accessibility and encourages the adoption of more advanced metrics. Melodia is a Python library with a complete set of components devised for describing, comparing and analysing the shape of protein structures using differential geometry of three-dimensional curves and knot theory. It can generate robust geometric descriptors for thousands of shapes in just a few minutes. Those descriptors are more sensitive to structural feature variation than RMSD deviation. Melodia also incorporates sequence structural annotation and three-dimensional visualisations. AVAILABILITY AND IMPLEMENTATION: Melodia is an open-source Python library freely available on https://github.com/rwmontalvao/Melodia_py, along with interactive Jupyter Notebook tutorials. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

EPSP synthase flexibility is determinant to its function: computational molecular dynamics and metadynamics studies.

Flexibility is involved in a wide range of biological processes, such as protein assembly and binding recognition. EPSP synthase is an enzyme that must undergo a large conformational change to accommodate its ligands into its binding cavity. However, although the structure of EPSP synthase has been determined, its plasticity has not been explored in depth. Therefore, in this work, we extensively examined the influence of the flexibility of Mycobacterium tuberculosis EPSP (MtEPSP) synthase on the function of this protein using classical and replica-exchange metadynamics simulations. We were able to identify five well-populated conformational clusters for MtEPSP synthase: two corresponding to open, one to ajar, and two to closed conformations. We also pinpointed three hydrophobic regions that are responsible for guiding transitions among these states. Taken together, the new findings presented here indicate how the hydrophobic regions modulate the flexibility of MtEPSP synthase, and they highlight the importance of considering these dynamic features in drug design projects employing this enzyme as a target. Graphical abstract The flexibility of EPSP synthase as a function of the pincer angles.

Polyphony: superposition independent methods for ensemble-based drug discovery.

BACKGROUND: Structure-based drug design is an iterative process, following cycles of structural biology, computer-aided design, synthetic chemistry and bioassay. In favorable circumstances, this process can lead to the structures of hundreds of protein-ligand crystal structures. In addition, molecular dynamics simulations are increasingly being used to further explore the conformational landscape of these complexes. Currently, methods capable of the analysis of ensembles of crystal structures and MD trajectories are limited and usually rely upon least squares superposition of coordinates. RESULTS: Novel methodologies are described for the analysis of multiple structures of a protein. Statistical approaches that rely upon residue equivalence, but not superposition, are developed. Tasks that can be performed include the identification of hinge regions, allosteric conformational changes and transient binding sites. The approaches are tested on crystal structures of CDK2 and other CMGC protein kinases and a simulation of p38α. Known interaction - conformational change relationships are highlighted but also new ones are revealed. A transient but druggable allosteric pocket in CDK2 is predicted to occur under the CMGC insert. Furthermore, an evolutionarily-conserved conformational link from the location of this pocket, via the αEF-αF loop, to phosphorylation sites on the activation loop is discovered. CONCLUSIONS: New methodologies are described and validated for the superimposition independent conformational analysis of large collections of structures or simulation snapshots of the same protein. The methodologies are encoded in a Python package called Polyphony, which is released as open source to accompany this paper [http://wrpitt.bitbucket.org/polyphony/].

Structures of the excited states of phospholamban and shifts in their populations upon phosphorylation.

Phospholamban is an integral membrane protein that controls the calcium balance in cardiac muscle cells. As the function and regulation of this protein require the active involvement of low populated states in equilibrium with the native state, it is of great interest to acquire structural information about them. In this work, we calculate the conformations and populations of the ground state and the three main excited states of phospholamban by incorporating nuclear magnetic resonance residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations. We then provide a description of the manner in which phosphorylation at Ser16 modulates the activity of the protein by increasing the sizes of the populations of its excited states. These results demonstrate that the approach that we describe provides a detailed characterization of the different states of phospholamban that determine the function and regulation of this membrane protein. We anticipate that the knowledge of conformational ensembles enable the design of new dominant negative mutants of phospholamban by modulating the relative populations of its conformational substates.

Characterization of the interdomain motions in hen lysozyme using residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations.

Hen lysozyme is an enzyme characterized by the presence of two domains whose relative motions are involved in the mechanism of binding and release of the substrates. By using residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations, we characterize the breathing motions describing the interdomain fluctuations of this protein. We found that the ensemble of conformations that we determined spans the entire range of structures of hen lysozyme deposited in the Protein Data Bank, including both the free and bound states, suggesting that the thermal motions in the free state provide access to the structures populated upon binding. The approach that we present illustrates how the use of residual dipolar couplings as replica-averaged structural restraints in molecular dynamics simulations makes it possible to explore conformational fluctuations of a relatively large amplitude in proteins.

A method of determining RNA conformational ensembles using structure-based calculations of residual dipolar couplings.

We describe a method of determining the conformational fluctuations of RNA based on the incorporation of nuclear magnetic resonance (NMR) residual dipolar couplings (RDCs) as replica-averaged structural restraints in molecular dynamics simulations. In this approach, the alignment tensor required to calculate the RDCs corresponding to a given conformation is estimated from its shape, and multiple replicas of the RNA molecule are simulated simultaneously to reproduce in silico the ensemble-averaging procedure performed in the NMR measurements. We provide initial evidence that with this approach it is possible to determine accurately structural ensembles representing the conformational fluctuations of RNA by applying the reference ensemble test to the trans-activation response element of the human immunodeficiency virus type 1.

Determination of structural fluctuations of proteins from structure-based calculations of residual dipolar couplings.

Residual dipolar couplings (RDCs) have the potential of providing detailed information about the conformational fluctuations of proteins. It is very challenging, however, to extract such information because of the complex relationship between RDCs and protein structures. A promising approach to decode this relationship involves structure-based calculations of the alignment tensors of protein conformations. By implementing this strategy to generate structural restraints in molecular dynamics simulations we show that it is possible to extract effectively the information provided by RDCs about the conformational fluctuations in the native states of proteins. The approach that we present can be used in a wide range of alignment media, including Pf1, charged bicelles and gels. The accuracy of the method is demonstrated by the analysis of the Q factors for RDCs not used as restraints in the calculations, which are significantly lower than those corresponding to existing high-resolution structures and structural ensembles, hence showing that we capture effectively the contributions to RDCs from conformational fluctuations.

Determination of Conformational Equilibria in Proteins Using Residual Dipolar Couplings.

In order to carry out their functions, proteins often undergo significant conformational fluctuations that enable them to interact with their partners. The accurate characterization of these motions is key in order to understand the mechanisms by which macromolecular recognition events take place. Nuclear magnetic resonance spectroscopy offers a variety of powerful methods to achieve this result. We discuss a method of using residual dipolar couplings as replica-averaged restraints in molecular dynamics simulations to determine large amplitude motions of proteins, including those involved in the conformational equilibria that are established through interconversions between different states. By applying this method to ribonuclease A, we show that it enables one to characterize the ample fluctuations in interdomain orientations expected to play an important functional role.

Using chemical shifts to determine structural changes in proteins upon complex formation.

Methods for determining protein structures using only chemical shift information are progressively becoming more accurate and reliable. A major problem, however, in the use of chemical shifts for the determination of the structures of protein complexes is that the changes in the chemical shifts upon binding tend to be rather limited and indeed often smaller than the standard errors made in the predictions of chemical shifts corresponding to given structures. We present a procedure that, despite this problem, enables one to use of chemical shifts to determine accurately the conformational changes that take place upon complex formation.

Structure determination of protein-protein complexes using NMR chemical shifts: case of an endonuclease colicin-immunity protein complex.

Nuclear magnetic resonance (NMR) spectroscopy provides a range of powerful techniques for determining the structures and the dynamics of proteins. The high-resolution determination of the structures of protein-protein complexes, however, is still a challenging problem for this approach, since it can normally provide only a limited amount of structural information at protein-protein interfaces. We present here the determination using NMR chemical shifts of the structure (PDB code 2K5X) of the cytotoxic endonuclease domain from bacterial toxin colicin (E9) in complex with its cognate immunity protein (Im9). In order to achieve this result, we introduce the CamDock method, which combines a flexible docking procedure with a refinement that exploits the structural information provided by chemical shifts. The results that we report thus indicate that chemical shifts can be used as structural restraints for the determination of the conformations of protein complexes that are difficult to obtain by more standard NMR approaches.

Andante: reducing side-chain rotamer search space during comparative modeling using environment-specific substitution probabilities.

MOTIVATION: The accurate placement of side chains in computational protein modeling and design involves the searching of vast numbers of rotamer combinations. RESULTS: We have applied the information contained within structurally aligned homologous families, in the form of conserved chi angle conservation rules, to the problem of the comparative modeling. This allows the accurate borrowing of entire side-chain conformations and/or the restriction to high probability rotamer bins. The application of these rules consistently reduces the number of rotamer combinations that need to be searched to trivial values and also reduces the overall side-chain root mean square deviation (rmsd) of the final model. The approach is complementary to current side-chain placement algorithms that use the decomposition of interacting clusters to increase the speed of the placement process.

CHORAL: a differential geometry approach to the prediction of the cores of protein structures.

MOTIVATION: Although the cores of homologous proteins are relatively well conserved, amino acid substitutions lead to significant differences in the structures of divergent superfamilies. Thus, the classification of amino acid sequence patterns and the selection of appropriate fragments of the protein cores of homologues of known structure are important for accurate comparative modelling. RESULTS: CHORAL utilizes a knowledge-based method comprising an amalgam of differential geometry and pattern recognition algorithms to identify conserved structural patterns in homologous protein families. Propensity tables are used to classify and to select patterns that most likely represent the structure of the core for a target protein. In our benchmark, CHORAL demonstrates a performance equivalent to that of MODELLER.