Towards more robust and efficient methods for the calculation of Protein-Ligand binding affinities

Biological processes often depend on protein-ligand binding events, so that accurate prediction of protein-ligand binding affinities is of central importance in structural based drug design. Although many techniques exist for calculating protein-ligand binding affinities, ranging from techniques that should be accurate in principle, such as free energy perturbation (FEP) theory, to relatively simple approximations based on empirically derived scoring functions, the counterbalancing demands of speed and accuracy have left us with no completely satisfactory solution thus far. This thesis will be focused on the methodology development towards more robust and reliable Protein-Ligand binding affinity calculation. In Part I, we will present the WaterMap method, which will bridge the gap between the efficiency of empirical scoring functions and the accuracy of rigorous FEP methods. Unlike most other methods with the main focus on the direct interaction between the protein and the ligand, the WaterMap method we developed considers the explicit driving force from the solvent, in which several individual water molecules in the binding pocket play an active role in the binding process. We demonstrate that protein may adopt active site geometries that will destabilize the water molecules in the binding pocket through hydrophobic enclosure and/or correlated hydrogen bonds, and displacement of these water molecules by ligand groups complementary to protein surface will provide the driving force for ligand binding. In some extreme cases, the interactions are so unfavorable for water molecules that a void is formed in the binding pocket of protein. Our method also considers the contribution from occupation of ligand atoms in the dry regions of binding pocket, which in some cases provides the driving force for ligand binding. FEP provides an in-principle rigorous method to calculate protein-ligand binding affinities within the limitations of the potential energy model and it may have a potentially large impact on structure based drug design projects especially during late stage lead optimization when productive decisions about compound modification are made . However, converging explicit solvent simulations to the desired precision is far from trivial, especially when there are large structural reorganizations in the protein or in the ligand upon the formation of the binding complex or upon the alchemical transformation from one ligand to another. In these cases, there can be large energy barriers separating the different conformations and the ligand or the protein may remain kinetically trapped in the starting configuration for a very long time during brute-force FEP/MD simulations. The incomplete sampling of the configuration space results in the computed binding free energies being dependent on the starting protein or ligand configurations, thus giving rise to the well known quasi-nonergodicity problem in FEP. In Part II, we will present a new protocol called FEP/REST, which combines the recently developed enhanced sampling technique REST (Replica Exchange with Solute Tempering) into normal FEP to solve the sampling problem in brute force FEP calculation. The computational cost of this method is comparable with normal FEP, and it can be very easily generalized to more complicated systems of pharmaceutical interest. We apply this method to two modifications of protein-ligand complexes which lead to significant conformational changes, the first in the protein and the second in the ligand. The new approach is shown to facilitate sampling in these challenging cases where high free energy barriers separate the initial and final conformations, and leads to superior convergence of the free energy as demonstrated both by consistency of the results (independence from the starting conformation) and agreement with experimental binding affinity data. Part III focus on two topics towards the foundational understanding of hydrophobic interactions and electrostatic interactions. To be specific, the nonadditivity effect of hydrophobic interactions in model enclosures is studied in Chapter 9, and the competition between hydrophobic interaction and electrostatic interaction between a hydrophobe and model enclosure is studied in Chapter 10. The approximations in popular implicit solvent models, like the surface area model in hydrophobic interaction, and the quadratic dependence of electrostatic interaction on the magnitude of charge are investigated. Six of the Chapters (Chapter 2-4, Chapter 6, and Chapter 9-10) have been published and the other one (Chapter 7) has been accepted for publication and currently is in press. Each Part begins with its own introduction. Each chapter also contains its own abstract and introduction, and focus on one specific topic. They all share the common theme, that is to develop more robust and reliable methods to calculate protein-ligand binding affinities. The conclusions and discussions about future research directions are presented in Part IV

N-terminal Inactivation Domains of β Subunits Are Protected from Trypsin Digestion by Binding within the Antechamber of BK Channels

N termini of auxiliary β subunits that produce inactivation of large-conductance Ca2+-activated K+ (BK) channels reach their pore-blocking position by first passing through side portals into an antechamber separating the BK pore module and the large C-terminal cytosolic domain. Previous work indicated that the β2 subunit inactivation domain is protected from digestion by trypsin when bound in the inactivated conformation. Other results suggest that, even when channels are closed, an inactivation domain can also be protected from digestion by trypsin when bound within the antechamber. Here, we provide additional tests of this model and examine its applicability to other β subunit N termini. First, we show that specific mutations in the β2 inactivation segment can speed up digestion by trypsin under closed-channel conditions, supporting the idea that the β2 N terminus is protected by binding within the antechamber. Second, we show that cytosolic channel blockers distinguish between protection mediated by inactivation and protection under closed-channel conditions, implicating two distinct sites of protection. Together, these results confirm the idea that β2 N termini can occupy the BK channel antechamber by interaction at some site distinct from the BK central cavity. In contrast, the β3a N terminus is digested over 10-fold more quickly than the β2 N terminus. Analysis of factors that contribute to differences in digestion rates suggests that binding of an N terminus within the antechamber constrains the trypsin accessibility of digestible basic residues, even when such residues are positioned outside the antechamber. Our analysis indicates that up to two N termini may simultaneously be protected from digestion. These results indicate that inactivation domains have sites of binding in addition to those directly involved in inactivation

Knockout of the BK β2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four β2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of β2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing β2 subunits, we generated mice in which the gene encoding β2 was deleted (β2 knockout [KO]). Comparison of proteins from wild-type (WT) and β2 KO mice allowed unambiguous demonstration of the presence of β2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and β2 KO CCs in slices and found that β2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the β2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from β2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of β2 subunits may predispose to burst firing

Control of Centrin Stability by Aurora A

Aurora A is an oncogenic serine/threonine kinase which can cause cell transformation and centrosome amplification when over-expressed. Human breast tumors show excess Aurora A and phospho-centrin in amplified centrosomes. Here, we show that Aurora A mediates the phosphorylation of and localizes with centrin at the centrosome, with both proteins reaching maximum abundance from prophase through metaphase, followed by their precipitous loss in late stages of mitosis. Over-expression of Aurora A results in excess phospho-centrin and centrosome amplification. In contrast, centrosome amplification is not seen in cells over-expressing Aurora A in the presence of a recombinant centrin mutant lacking the serine phosphorylation site at residue 170. Expression of a kinase dead Aurora A results in a decrease in mitotic index and abrogation of centrin phosphorylation. Finally, a recombinant centrin mutation that mimics centrin phosphorylation increases centrin's stability against APC/C-mediated proteasomal degradation. Taken together, these results suggest that the stability of centrin is regulated in part by Aurora A, and that excess phosphorylated centrin may promote centrosome amplification in cancer

The RCK1 domain of the human BK_(Ca) channel transduces Ca^(2+) binding into structural rearrangements

Large-conductance voltage- and Ca^(2+)-activated K^+ (BK_(Ca)) channels play a fundamental role in cellular function by integrating information from their voltage and Ca2+ sensors to control membrane potential and Ca^(2+) homeostasis. The molecular mechanism of Ca^(2+)-dependent regulation of BKCa channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K^+ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BK_(Ca) RCK1 domain adopts an α/β fold, binds Ca^(2+), and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca^(2+)-induced conformational changes in physiologically relevant [Ca^(2+)]. The neutralization of residues known to be involved in high-affinity Ca^(2+) sensing (D362 and D367) prevented Ca^(2+)-induced structural transitions in RCK1 but did not abolish Ca^(2+) binding. We provide evidence that the RCK1 domain is a high-affinity Ca^(2+) sensor that transduces Ca^(2+) binding into structural rearrangements, likely representing elementary steps in the Ca^(2+)-dependent activation of human BK_(Ca) channels

Paxilline inhibits BK channels by an almost exclusively closed-channel block mechanism

Paxilline, a tremorogenic fungal alkaloid, potently inhibits large conductance Ca(2+)- and voltage-activated K(+) (BK)-type channels, but little is known about the mechanism underlying this inhibition. Here we show that inhibition is inversely dependent on BK channel open probability (Po), and is fully relieved by conditions that increase Po, even in the constant presence of paxilline. Manipulations that shift BK gating to more negative potentials reduce inhibition by paxilline in accordance with the increase in channel Po. Measurements of Po times the number of channels at negative potentials support the idea that paxilline increases occupancy of closed states, effectively reducing the closed–open equilibrium constant, L(0). Gating current measurements exclude an effect of paxilline on voltage sensors. Steady-state inhibition by multiple paxilline concentrations was determined for four distinct equilibration conditions, each with a distinct Po. The IC(50) for paxilline shifted from around 10 nM when channels were largely closed to near 10 µM as maximal Po was approached. Model-dependent analysis suggests a mechanism of inhibition in which binding of a single paxilline molecule allosterically alters the intrinsic L(0) favoring occupancy of closed states, with affinity for the closed conformation being >500-fold greater than affinity for the open conformation. The rate of inhibition of closed channels was linear up through 2 µM paxilline, with a slope of 2 × 10(6) M(−1)s(−1). Paxilline inhibition was hindered by either the bulky cytosolic blocker, bbTBA, or by concentrations of cytosolic sucrose that hinder ion permeation. However, paxilline does not hinder MTSET modification of the inner cavity residue, A313C. We conclude that paxilline binds more tightly to the closed conformation, favoring occupancy of closed-channel conformations, and propose that it binds to a superficial position near the entrance to the central cavity, but does not hinder access of smaller molecules to this cavity

PLK1 Interacts and Phosphorylates Axin That Is Essential for Proper Centrosome Formation

10.1371/journal.pone.0049184PLoS ONE711

Metal-driven Operation of the Human Large-conductance Voltage- and Ca^(2+)-dependent Potassium Channel (BK) Gating Ring Apparatus

Large-conductance voltage- and Ca^(2+)-dependent K^+ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca^(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the “gating ring”), which confers sensitivity to intracellular Ca^(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca^(2+)- and Mg^(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba^(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca^(2+)-binding site (the “calcium bowl”) reduced the Ca^(2+) and abolished the Mg^(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca^(2+)-binding site and that Mg^(2+) can bind to the calcium bowl with less affinity than Ca^(2+). Dynamic light scattering analysis revealed a reversible Ca^(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel

Quantitative miRNA Expression Analysis Using Fluidigm Microfluidics Dynamic Arrays

MicroRNA (miRNA) is a small non-coding RNA that can regulate gene expression in both plants and animals. Studies showed that miRNAs play a critical role in human cancer by targeting messenger RNAs that are positive or negative regulators of cell proliferation and apoptosis. Here, we evaluated miRNA expression in formalin fixed, paraffin embedded (FFPE) samples and fresh frozen (FF) samples using a high throughput qPCR-based microfluidic dynamic array technology (Fluidigm). We compared the results to hybridization-based microarray platforms using the same samples. We obtained a highly correlated Ct values between multiplex and single-plex RT reactions using standard qPCR assays for miRNA expression. For the same samples, the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left shift towards lower Ct values compared to those observed by standard TaqMan (ABI 7900HT, mean difference, 3.79). In addition, as little as 10ng total RNA was sufficient to reproducibly detect up to 96 miRNAs at a wide range of expression values using a single 96-multiplexing RT reaction in either FFPE or FF samples. Comparison of miRNAs expression values measured by microfluidic technology with those obtained by other array and Next Generation sequencing platforms showed positive concordance using the same samples but revealed significant differences for a large fraction of miRNA targets. The qPCRarray based microfluidic technology can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. This approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform while achieving much higher throughput at lower sample input and reagent usage. It is a rapid, cost effective, customizable array platform for miRNA expression profiling and validation. However, comparison of miRNA expression using different platforms requires caution and the use of multiple platforms