Sirish Kaushik Lakkaraju

Pharmacophore modeling using site-identification by ligand competitive saturation (SILCS) with multiple probe molecules.

Receptor-based pharmacophore modeling is an efficient computer-aided drug design technique that uses the structure of the target protein to identify novel leads. However, most methods consider protein flexibility and desolvation effects in a very approximate way, which may limit their use in practice. The Site-Identification by Ligand Competitive Saturation (SILCS) assisted pharmacophore modeling protocol (SILCS-Pharm) was introduced recently to address these issues, as SILCS naturally takes both protein flexibility and desolvation effects into account by using full molecular dynamics simulations to determine 3D maps of the functional group-affinity patterns on a target receptor. In the present work, the SILCS-Pharm protocol is extended to use a wider range of probe molecules including benzene, propane, methanol, formamide, acetaldehyde, methylammonium, acetate and water. This approach removes the previous ambiguity brought by using water as both the hydrogen-bond donor and acceptor probe molecule. The new SILCS-Pharm protocol is shown to yield improved screening results, as compared to the previous approach based on three target proteins. Further validation of the new protocol using five additional protein targets showed improved screening compared to those using common docking methods, further indicating improvements brought by the explicit inclusion of additional feature types associated with the wider collection of probe molecules in the SILCS simulations. The advantage of using complementary features and volume constraints, based on exclusion maps of the protein defined from the SILCS simulations, is presented. In addition, reranking using SILCS-based ligand grid free energies is shown to enhance the diversity of identified ligands for the majority of targets. These results suggest that the SILCS-Pharm protocol will be of utility in rational drug design.

Hysteresis-based mechanism for the directed motility of the Ncd motor.

Ncd is a Kinesin-14 family protein that walks to the microtubules minus end. Although available structures show its α-helical neck in either pre- or post-stroke orientations, little is known about the transition between these two states. Using a combination of molecular dynamics simulations and structural analyses, we find that the neck sequentially makes intermediate contacts with the motor head along its mostly longitudinal path, and it develops a 24° twist in the post-stroke orientation. The forward (pre-stroke to post-stroke) motion has an ∼4.5 k(B)T (where k(B) is the Boltzmann constant, and T=300 K) free-energy barrier and is a diffusion guided by the intermediate contacts. The post-stroke free-energy minimum is higher and is formed ∼10° before reaching the orientation in the post-stroke crystal structure, consistent with previous structural data. The importance of intermediate contacts correlates with the existing motility data, including those for mutant Ncds. Unlike the forward motion, the recovery stroke goes nearly downhill in free energy, powered in part by torsional relaxation of the neck. The hysteresis in the energetics of the neck motion arises from the mechanical compliance of the protein, and together with guided diffusion, it may be key to the directed motility of Ncd.

Characterization of Mg2+ Distributions around RNA in Solution

Binding of metal ions is an important factor governing the folding and dynamics of RNA. Shielding of charges in the polyanionic backbone allows RNA to adopt a diverse range of folded structures that give rise to their many functions within the cell. Some RNA sequences fold only in the presence of Mg2+, which may be bound via direct interactions or occupy the more diffuse “ion atmosphere” around the RNA. To understand the driving forces for RNA folding, it is important to be able to fully characterize the distribution of metal ions around the RNA. In this work, a combined Grand Canonical Monte Carlo-Molecular Dynamics (GCMC-MD) method is applied to characterize Mg2+ distributions around folded RNA structures. The GCMC-MD approach identifies known inner- and outer-shell Mg2+ coordination, while also predicting new regions occupied by Mg2+ that are not observed in crystal structures but that may be relevant in solution, including the case of the Mg2+ riboswitch, for which alternate Mg2+ binding sites may have implications for its function. This work represents a significant step forward in establishing a structural and thermodynamic description of RNA–Mg2+ interactions and their role in RNA structure and function.

Determination of Ionic Hydration Free Energies with Grand Canonical Monte Carlo/Molecular Dynamics Simulations in Explicit Water

Grand canonical Monte Carlo (GCMC) simulations of ionic solutions with explicit solvent models are known to be challenging. One challenge arises from the treatment of long-range electrostatics and finite-box size in Monte Carlo simulations when periodic boundary condition and Ewald summation methods are used. Another challenge is that constant excess chemical potential GCMC simulations for charged solutes suffer from inadequate insertion and deletion acceptance ratios. In this work, we address those problems by implementing an oscillating excess chemical potential GCMC algorithm with smooth particle mesh Ewald and finite-box-size corrections to treat the long-range electrostatics. The developed GCMC simulation program was combined with GROMACS to perform GCMC/MD simulations of ionic solutions individually containing Li+, Na+, K+, Rb+, Cs+, F-, Cl-, Br-, I-, Ca2+, and Mg2+, respectively. Our simulation results show that the combined GCMC/MD approach can approximate the ionic hydration free energies with proper treatment of long-range electrostatics. Our developed simulation approach can open up new avenues for simulating complex chemical and biomolecular systems and for drug discovery.

Transition Pathway for the Minus-End Directed Movement of Kinesin-14 NCD

Using Targeted Molecular Dynamics (TMD), we trace the transition pathway of the minus-end directed movement of the Ncd stalk. TMD reveals a number of substeps involved in the rotation of the stalk towards the minus end of microtubules (MT). Our results show that the stalk motion does not possess any directional bias (neither towards the plus or minus end of MT). The minus-end directed movement could thus be due to thermal diffusion between the series of substeps, rather than a rigid body rotation driven by a power stroke. Further tests are necessary to characterize substeps and the relative role played by thermal fluctuation and deterministic motion.Copyright

Length and Sequence Dependence of the Elasticity of Alpha Helices and Coiled-Coils

Fibrous proteins made by α-helices, one of the most elementary protein secondary structures, have various mechanical roles in a sub cellular environment. An α-helix is wound in a right-handed fashion due to hydrogen bonding between the C=O and the N-H atoms across every i and i+4th residues in the polypeptide chain. Previous approaches characterizing mechanical properties of α-helices treated them as homogenous and linear elastic rods. Stiffness is typically expressed in terms of persistence length lp (∼100nm from Kb∼3×10−28 Nm2, lp = Kb/kT: Kb, the bending stiffness, k the Boltzmann constant and T = 300 K, the temperature) [1–3]. In this study, we show that bending stiffness depends on the length of the filament, due to inherent non-bonded attractions. In particular, non-bonded attraction introduces a new length scale, critical buckling length, beyond which the filament can no longer remain linearly elastic. These results suggest that non-bonded attractions can significantly affect elastic properties of biofilament systems such as the cytoskeleton. Furthermore, we find that while elasticity of a single α-helix is largely independent of its amino acid sequence, α-helical coiled-coils have stronger sequence dependence, and in the case of tropomyosin molecule, we find regional variations in the flexibility which may have functional implications in its actin binding properties and muscle contraction.Copyright