David Sept

Electrostatics of nanosystems: Application to microtubules and the ribosome

Evaluation of the electrostatic properties of biomolecules has become a standard practice in molecular biophysics. Foremost among the models used to elucidate the electrostatic potential is the Poisson-Boltzmann equation; however, existing methods for solving this equation have limited the scope of accurate electrostatic calculations to relatively small biomolecular systems. Here we present the application of numerical methods to enable the trivially parallel solution of the Poisson-Boltzmann equation for supramolecular structures that are orders of magnitude larger in size. As a demonstration of this methodology, electrostatic potentials have been calculated for large microtubule and ribosome structures. The results point to the likely role of electrostatics in a variety of activities of these structures.

Thermodynamics and kinetics of actin filament nucleation.

We have performed computer simulations and free energy calculations to determine the thermodynamics and kinetics of actin nucleation and thus identify a probable nucleation pathway and critical nucleus size. The binding free energies of structures along the nucleation pathway are found through a combination of electrostatic calculations and estimates of the entropic and surface area contributions. The association kinetics for the formation of each structure are determined through a series of Brownian dynamics simulations. The combination of the binding free energies and the association rate constants determines the dissociation rate constants, allowing for a complete characterization of the nucleation and polymerization kinetics. The results indicate that the trimer is the size of the critical nucleus, and the rate constants produce polymerization plots that agree very well with experimental results over a range of actin monomer concentrations.

The physical basis of microtubule structure and stability

Microtubules are cylindrical polymers found in every eukaryotic cell. They have a unique helical structure that has implications at both the cellular level, in terms of the functions they perform, and at the multicellular level, such as determining the left–right symmetry in plants. Through the combination of an atomically detailed model for a microtubule and large‐scale computational techniques for computing electrostatic interactions, we are able to explain the observed microtubule structure. On the basis of the lateral interactions between protofilaments, we have determined that B lattice is the most favorable configuration. Further, we find that these lateral bonds are significantly weaker than the longitudinal bonds along protofilaments. This explains observations of microtubule disassembly and may serve as another step toward understanding the basis for dynamic instability.

A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB.

S-(3,4-Dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homologue MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We show that A22 binds MreB with at least micromolar affinity in its nucleotide-binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly properties that more closely resemble those of the ADP-bound state. Because the cellular concentration of MreB is in the low micromolar range, this mechanism explains the ability of A22 to largely disassemble the actin cytoskeleton in bacterial cells. It also represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.

The adaptive multilevel finite element solution of the Poisson-Boltzmann equation on massively parallel computers

By using new methods for the parallel solution of elliptic partial differential equations, the teraflops computing power of massively parallel computers can be leveraged to perform electrostatic calculations on large biological systems. This paper describes the adaptive multilevel finite element solution of the Poisson-Boltzmann equation for a microtubule on the NPACI Blue Horizon--a massively parallel IBM RS/6000® SP with eight POWER3 SMP nodes. The microtubule system is 40 nm in length and 24 nm in diameter, consists of roughly 600000 atoms, and has a net charge of -1800 e. Poisson-Boltzmann calculations are performed for several processor configurations, and the algorithm used shows excellent parallel scaling.

A multiscale model linking ion-channel molecular dynamics and electrostatics to the cardiac action potential

Ion-channel function is determined by its gating movement. Yet, molecular dynamics and electrophysiological simulations were never combined to link molecular structure to function. We performed multiscale molecular dynamics and continuum electrostatics calculations to simulate a cardiac K+ channel (IKs) gating and its alteration by mutations that cause arrhythmias and sudden death. An all-atom model of the IKs α-subunit KCNQ1, based on the recent Kv1.2 structure, is used to calculate electrostatic energies during gating. Simulations are compared with experiments where varying degrees of positive charge—added via point mutation—progressively reduce current. Whole-cell simulations show that mutations cause action potential and ECG QT interval prolongation, consistent with clinical phenotypes. This framework allows integration of multiscale observations to study the molecular basis of excitation and its alteration by disease.

Annealing Accounts for the Length of Actin Filaments Formed by Spontaneous Polymerization

We measured the lengths of actin filaments formed by spontaneous polymerization of highly purified actin monomers by fluorescence microscopy after labeling with rhodamine-phalloidin. The length distributions are exponential with a mean of approximately 7 microm (2600 subunits). This length is independent of the initial concentration of actin monomer, an observation inconsistent with a simple nucleation-elongation mechanism. However, with the addition of physically reasonable rates of filament annealing and fragmenting, a nucleation-elongation mechanism can reproduce the observed average length of filaments in two types of experiments: 1) filaments formed from a wide range of highly purified actin monomer concentrations, and 2) filaments formed from 24 microM actin over a range of CapZ concentrations.

Taxol Allosterically Alters the Dynamics of the Tubulin Dimer and Increases the Flexibility of Microtubules

Taxol is a commonly used antitumor agent that hyperstabilizes microtubules and prevents cell division. The interaction of Taxol with tubulin and the microtubule has been studied through a wide array of experimental techniques; however, the exact molecular mechanism by which Taxol stabilizes microtubules has remained elusive. In this study, through the use of large-scale molecular simulations, we show that Taxol affects the interactions between the M and H1-S2 loops of adjacent tubulin dimers leading to more stable interprotofilament interactions. More importantly, we demonstrate that Taxol binding leads to a significant increase in the dynamics and flexibility of the portion of beta-tubulin that surrounds the bound nucleotide and makes contact with the alpha-monomer of the next dimer in the protofilament. We conclude that this increase in flexibility allows the microtubule to counteract the conformational changes induced by nucleotide hydrolysis and keeps the protofilaments in a straight conformation, resulting in a stable microtubule.

Evolutionarily Divergent, Unstable Filamentous Actin Is Essential for Gliding Motility in Apicomplexan Parasites

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein IMPLICATIONS FOR HOW CAPPING PROTEIN BINDS THE ACTIN FILAMENT

The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP2). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP2 binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP2 binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP2 rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can “wobble” when bound to the barbed end solely by the C-terminal “tentacle” of its β-subunit.