Variational Methods for Biomolecular Modeling

Structure, function and dynamics of many biomolecular systems can be characterized by the energetic variational principle and the corresponding systems of partial differential equations (PDEs). This principle allows us to focus on the identification of essential energetic components, the optimal parametrization of energies, and the efficient computational implementation of energy variation or minimization. Given the fact that complex biomolecular systems are structurally non-uniform and their interactions occur through contact interfaces, their free energies are associated with various interfaces as well, such as solute-solvent interface, molecular binding interface, lipid domain interface, and membrane surfaces. This fact motivates the inclusion of interface geometry, particular its curvatures, to the parametrization of free energies. Applications of such interface geometry based energetic variational principles are illustrated through three concrete topics: the multiscale modeling of biomolecular electrostatics and solvation that includes the curvature energy of the molecular surface, the formation of microdomains on lipid membrane due to the geometric and molecular mechanics at the lipid interface, and the mean curvature driven protein localization on membrane surfaces. By further implicitly representing the interface using a phase field function over the entire domain, one can simulate the dynamics of the interface and the corresponding energy variation by evolving the phase field function, achieving significant reduction of the number of degrees of freedom and computational complexity. Strategies for improving the efficiency of computational implementations and for extending applications to coarse-graining or multiscale molecular simulations are outlined.Comment: 36 page

Characterizing Loop Dynamics and Ligand Recognition in Human- and Avian-Type Influenza Neuraminidases via Generalized Born Molecular Dynamics and End-Point Free Energy Calculations

The comparative dynamics and inhibitor binding free energies of group-1 and group-2 pathogenic influenza A subtype neuraminidase (NA) enzymes are of fundamental biological interest and relevant to structure-based drug design studies for antiviral compounds. In this work, we present seven generalized Born molecular dynamics simulations of avian (N1)- and human (N9)-type NAs in order to probe the comparative flexibility of the two subtypes, both with and without the inhibitor oseltamivir bound. The enhanced sampling obtained through the implicit solvent treatment suggests several provocative insights into the dynamics of the two subtypes, including that the group-2 enzymes may exhibit similar motion in the 430-binding site regions but different 150-loop motion. End-point free energy calculations elucidate the contributions to inhibitor binding free energies and suggest that entropic considerations cannot be neglected when comparing across the subtypes. We anticipate the findings presented here will have broad implications for the development of novel antiviral compounds against both seasonal and pandemic influenza strains

Prediction of hot spot residues at protein-protein interfaces by combining machine learning and energy-based methods

Background: Alanine scanning mutagenesis is a powerful experimental methodology for investigating the structural and energetic characteristics of protein complexes. Individual aminoacids are systematically mutated to alanine and changes in free energy of binding (Delta Delta G) measured. Several experiments have shown that protein-protein interactions are critically dependent on just a few residues ("hot spots") at the interface. Hot spots make a dominant contribution to the free energy of binding and if mutated they can disrupt the interaction. As mutagenesis studies require significant experimental efforts, there is a need for accurate and reliable computational methods. Such methods would also add to our understanding of the determinants of affinity and specificity in protein-protein recognition.Results: We present a novel computational strategy to identify hot spot residues, given the structure of a complex. We consider the basic energetic terms that contribute to hot spot interactions, i.e. van der Waals potentials, solvation energy, hydrogen bonds and Coulomb electrostatics. We treat them as input features and use machine learning algorithms such as Support Vector Machines and Gaussian Processes to optimally combine and integrate them, based on a set of training examples of alanine mutations. We show that our approach is effective in predicting hot spots and it compares favourably to other available methods. In particular we find the best performances using Transductive Support Vector Machines, a semi-supervised learning scheme. When hot spots are defined as those residues for which Delta Delta G >= 2 kcal/mol, our method achieves a precision and a recall respectively of 56% and 65%.Conclusion: We have developed an hybrid scheme in which energy terms are used as input features of machine learning models. This strategy combines the strengths of machine learning and energy-based methods. Although so far these two types of approaches have mainly been applied separately to biomolecular problems, the results of our investigation indicate that there are substantial benefits to be gained by their integration

Binding Modes of Peptidomimetics Designed to Inhibit STAT3

STAT3 is a transcription factor that has been found to be constitutively activated in a number of human cancers. Dimerization of STAT3 via its SH2 domain and the subsequent translocation of the dimer to the nucleus leads to transcription of anti-apoptotic genes. Prevention of the dimerization is thus an attractive strategy for inhibiting the activity of STAT3. Phosphotyrosine-based peptidomimetic inhibitors, which mimic pTyr-Xaa-Yaa-Gln motif and have strong to weak binding affinities, have been previously investigated. It is well-known that structures of protein-inhibitor complexes are important for understanding the binding interactions and designing stronger inhibitors. Experimental structures of inhibitors bound to the SH2 domain of STAT3 are, however, unavailable. In this paper we describe a computational study that combined molecular docking and molecular dynamics to model structures of 12 peptidomimetic inhibitors bound to the SH2 domain of STAT3. A detailed analysis of the modeled structures was performed to evaluate the characteristics of the binding interactions. We also estimated the binding affinities of the inhibitors by combining MMPB/GBSA-based energies and entropic cost of binding. The estimated affinities correlate strongly with the experimentally obtained affinities. Modeling results show binding modes that are consistent with limited previous modeling studies on binding interactions involving the SH2 domain and phosphotyrosine(pTyr)-based inhibitors. We also discovered a stable novel binding mode that involves deformation of two loops of the SH2 domain that subsequently bury the C-terminal end of one of the stronger inhibitors. The novel binding mode could prove useful for developing more potent inhibitors aimed at preventing dimerization of cancer target protein STAT3

Investigation of the Interaction between the Large and Small Subunits of Potato ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield

Matrix metalloproteinases in a sea urchin ligament with adaptable mechanical properties

Mutable collagenous tissues (MCTs) of echinoderms show reversible changes in tensile properties (mutability) that are initiated and modulated by the nervous system via the activities of cells known as juxtaligamental cells. The molecular mechanism underpinning this mechanical adaptability has still to be elucidated. Adaptable connective tissues are also present in mammals, most notably in the uterine cervix, in which changes in stiffness result partly from changes in the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). There have been no attempts to assess the potential involvement of MMPs in the echinoderm mutability phenomenon, apart from studies dealing with a process whose relationship to the latter is uncertain. In this investigation we used the compass depressor ligaments (CDLs) of the sea-urchin Paracentrotus lividus. The effect of a synthetic MMP inhibitor - galardin - on the biomechanical properties of CDLs in different mechanical states ("standard", "compliant" and "stiff") was evaluated by dynamic mechanical analysis, and the presence of MMPs in normal and galardin-treated CDLs was determined semi-quantitatively by gelatin zymography. Galardin reversibly increased the stiffness and storage modulus of CDLs in all three states, although its effect was significantly lower in stiff than in standard or compliant CDLs. Gelatin zymography revealed a progressive increase in total gelatinolytic activity between the compliant, standard and stiff states, which was possibly due primarily to higher molecular weight components resulting from the inhibition and degradation of MMPs. Galardin caused no change in the gelatinolytic activity of stiff CDLs, a pronounced and statistically significant reduction in that of standard CDLs, and a pronounced, but not statistically significant, reduction in that of compliant CDLs. Our results provide evidence that MMPs may contribute to the variable tensility of the CDLs, in the light of which we provide an updated hypothesis for the regulatory mechanism controlling MCT mutability

Unprocessed Viral DNA Could Be the Primary Target of the HIV-1 Integrase Inhibitor Raltegravir

Integration of HIV DNA into host chromosome requires a 3′-processing (3′-P) and a strand transfer (ST) reactions catalyzed by virus integrase (IN). Raltegravir (RAL), commonly used in AIDS therapy, belongs to the family of IN ST inhibitors (INSTIs) acting on IN-viral DNA complexes (intasomes). However, studies show that RAL fails to bind IN alone, but nothing has been reported on the behaviour of RAL toward free viral DNA. Here, we assessed whether free viral DNA could be a primary target for RAL, assuming that the DNA molecule is a receptor for a huge number of pharmacological agents. Optical spectroscopy, molecular dynamics and free energy calculations, showed that RAL is a tight binder of both processed and unprocessed LTR (long terminal repeat) ends. Complex formation involved mainly van der Waals forces and was enthalpy driven. Dissociation constants (Kds) revealed that RAL affinity for unbound LTRs was stronger than for bound LTRs. Moreover, Kd value for binding of RAL to LTRs and IC50 value (half concentration for inhibition) were in same range, suggesting that RAL binding to DNA and ST inhibition are correlated events. Accommodation of RAL into terminal base-pairs of unprocessed LTR is facilitated by an extensive end fraying that lowers the RAL binding energy barrier. The RAL binding entails a weak damping of fraying and correlatively of 3′-P inhibition. Noteworthy, present calculated RAL structures bound to free viral DNA resemble those found in RAL-intasome crystals, especially concerning the contacts between the fluorobenzyl group and the conserved 5′C4pA33′ step. We propose that RAL inhibits IN, in binding first unprocessed DNA. Similarly to anticancer drug poisons acting on topoisomerases, its interaction with DNA does not alter the cut, but blocks the subsequent joining reaction. We also speculate that INSTIs having viral DNA rather IN as main target could induce less resistance

Sequencing and Comparative Genome Analysis of Two Pathogenic Streptococcus gallolyticus Subspecies: Genome Plasticity, Adaptation and Virulence

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops