Yuk Y. Sham

Structured disorder and conformational selection.

Traditionally, molecular disorder has been viewed as local or global instability. Molecules or regions displaying disorder have been considered inherently unstructured. The term has been routinely applied to cases for which no atomic coordinates can be derived from crystallized molecules. Yet, even when it appears that the molecules are disordered, prevailing conformations exist, with population times higher than those of all alternate conformations. Disordered molecules are the outcome of rugged energy landscapes away from the native state around the bottom of the funnel. Ruggedness has a biological function, creating a distribution of structured conformers that bind via conformational selection, driving association and multimolecular complex formation, whether chain‐linked in folding or unlinked in binding. We classify disordered molecules into two types. The first type possesses a hydrophobic core. Here, even if the native conformation is unstable, it still has a large enough population time, enabling its experimental detection. In the second type, no such hydrophobic core exists. Hence, the native conformations of molecules belonging to this category have shorter population times, hindering their experimental detection. Although there is a continuum of distribution of hydrophobic cores in proteins, an empirical, statistically based hydrophobicity function may be used as a guideline for distinguishing the two disordered molecule types. Furthermore, the two types relate to steps in the protein folding reaction. With respect to protein design, this leads us to propose that engineering‐optimized specific electrostatic interactions to avoid electrostatic repulsion would reduce the type I disordered state, driving the molten globule (MG) → native (N) state. In contrast, for overcoming the type II disordered state, in addition to specific interactions, a stronger hydrophobic core is also indicated, leading to the denatured → MG → N state. Proteins 2001;44:418–427.

Examining methods for calculations of binding free energies: LRA, LIE, PDLD-LRA, and PDLD/S-LRA calculations of ligands binding to an HIV protease

Several strategies for evaluation of the protein‐ligand binding free energies are examined. Particular emphasis is placed on the Linear Response Approximation (LRA) (Lee et. al., Prot Eng 1992 ;5:215–228) and the Linear Interaction Energy (LIE) method (Åqvist et. al., Prot Eng 1994 ;7:385–391). The performance of the Protein Dipoles Langevin Dipoles (PDLD) method and its semi‐microscopic version (the PDLD/S method) is also considered. The examination is done by using these methods in the evaluating of the binding free energies of neutral C2‐symmetric cyclic urea‐based molecules to Human Immunodeficiency Virus (HIV) protease. Our starting point is the introduction of a thermodynamic cycle that decomposes the total binding free energy to electrostatic and non‐electrostatic contributions. This cycle is closely related to the cycle introduced in our original LRA study (Lee et. al., Prot Eng 1992 ;5:215–228). The electrostatic contribution is evaluated within the LRA formulation by averaging the protein‐ligand (and/or solvent‐ligand) electrostatic energy over trajectories that are propagated on the potentials of both the polar and non‐polar (where all residual charges are set to zero) states of the ligand. This average involves a scaling factor of 0.5 for the contributions from each state and this factor is being used in both the LRA and LIE methods. The difference is, however, that the LIE method neglects the contribution from trajectories over the potential of the non‐polar state. This approximation is entirely valid in studies of ligands in water but not necessarily in active sites of proteins. It is found in the present case that the contribution from the non‐polar states to the protein‐ligand binding energy is rather small. Nevertheless, it is clearly expected that this term is not negligible in cases where the protein provides preorganized environment to stabilize the residual charges of the ligand. This contribution can be particularly important in cases of charged ligands. The analysis of the non‐electrostatic term is much more complex. It is concluded that within the LRA method one has to complete the relevant thermodynamic cycle by evaluating the binding free energy of the “non‐polar” ligand, ℓ`, where all the residual charges are set to zero. It is shown that the LIE term, which involves the scaling of the van der Waals interaction by a constant β (usually in the order of 0.15 to 0.25), corresponds to this part of the cycle. In order to elucidate the nature of this non‐electrostatic term and the origin of the scaling constant β, it is important to evaluate explicitly the different contributions to the binding energy of the non‐polar ligand, ΔGbind,ℓ`. Since this cannot be done at present (for relatively large ligands) by rigorous free energy perturbation approaches, we evaluate ΔGbind,ℓ` by the PDLD approach, augmented by microscopic calculations of the change in configurational entropy upon binding. This evaluation takes into account the van der Waals, hydrophobic, water penetration and entropic contributions, which are the most important free energy contributions that make up the total ΔGbind,ℓ`. The sum of these contributions is scaled by a factor θ and it is argued that obtaining a quantitative balance between these contributions should result in θ = 1. By doing so we should have a reliable estimate of the value of the LIE β and a way to understand its origin. The present approach gives θ values between 0.5 and 0.73, depending on the approximation used. This is encouraging but still not satisfying. Nevertheless, one might be able to use our PDLD approach to estimate the change of the LIE θ between different protein active sites.

Direct Inhibition of Insulin-Like Growth Factor-I Receptor Kinase Activity by (−)−Epigallocatechin-3-Gallate Regulates Cell Transformation

Insulin-like growth factor-I receptor (IGF-IR) has been implicated in cancer pathophysiology. Furthermore, impairment of IGF-IR signaling in various cancer cell lines caused inhibition of the transformed phenotype as determined by the inhibition of colony formation in soft agar and the inhibition of tumor formation in athymic nude mice. Thus, the IGF-IR might be an attractive target for cancer prevention. We showed that the tea polyphenol, (−)−epigallocatechin-3-gallate (EGCG), is a small-molecule inhibitor of IGF-IR activity (IC50 of 14 μmol/L). EGCG abrogated anchorage-independent growth induced by IGF-IR overexpression and also prevented human breast and cervical cancer cell phenotype expression through inhibition of IGF-IR downstream signaling. Our findings are the first to show that the IGF-IR is a novel binding protein of EGCG and thus may help explain the chemopreventive effect of EGCG on cancer development. (Cancer Epidemiol Biomarkers Prev 2007;16(3):598–605)

Blue Matter, an application framework for molecular simulation on blue gene

In this paper we describe the context, architecture, and challenges of Blue Matter, the application framework being developed in conjunction with the science effort within IBMs Blue Gene project. The study of the mechanisms behind protein folding and related topics can require long time simulations on systems with a wide range of sizes and the application supporting these studies must map efficiently onto a large range of parallel partition sizes to optimize scientific throughput for a particular study. The design goals for the Blue Matter architecture include separating the complexities of the parallel implementation on a particular machine from those of the scientific simulation as well as minimizing system environmental dependencies so that running an application within a low overhead kernel with minimal services is possible. We describe some of the parallel decompositions currently being explored that target the first member of the Blue Gene family, BG/L, and present simple performance models for these decompositions that we are using to prioritize our development work. Preliminary results indicate that the high-performance networks on BG/L will allow us to use FFT-based techniques for periodic electrostatics with reasonable speedups on 512-1024 node count partitions even for systems with as few as 5000 atoms.

Simulating proton translocations in proteins: Probing proton transfer pathways in the Rhodobacter sphaeroides reaction center†

A general method for simulating proton translocations in proteins and for exploring the role of different proton transfer pathways is developed and examined. The method evaluates the rate constants for proton transfer processes using the energetics of the relevant proton configurations. The energies (ΔG(m)) of the different protonation states are evaluated in two steps. First, the semimicroscopic version of the protein dipole Langevin dipole (PDLD/S) method is used to evaluate the intrinsic energy of bringing the protons to their protein sites, when the charges of all protein ionized residues are set to zero. Second, the interactions between the charged groups are evaluated by using a Coulombs Law with an effective dielectric constant. This approach, which was introduced in an earlier study by one of the authors of the current report, allows for a very fast determination of any ΔG(m) and for practical evaluation of the time‐dependent proton population: That is, the rate constants for proton transfer processes are evaluated by using the corresponding ΔG(m) values and a Marcus type relationship. These rate constants are then used to construct a master equation, the integration of which by a fourth‐order Runge‐Kutta method yields the proton population as a function of time. The integration evaluates, ‘on the fly,’ the changes of the rate constants as a result of the time‐dependent changes in charge‐charge interaction, and this feature benefits from the fast determination of ΔG(m). The method is demonstrated in a preliminary study of proton translocation processes in the reaction center of Rhodobacter sphaeroides. It is found that proton transfer across water chains involves significant activation barriers and that ionized protein residues probably are involved in the proton transfer pathways. The potential of the present method in analyzing mutation experiments is discussed briefly and illustrated. The present study also examines different views of the nature of proton translocations in proteins. It is shown that such processes are controlled mainly by the electrostatic interaction between the proton site and its surroundings rather than by the local bond rearrangements of water molecules that are involved in the proton pathways. Thus, the overall rate of proton transport frequently is controlled by the highest barrier along the conduction pathway. Proteins 1999;36:484–500.

Rapid Identification of Keap1–Nrf2 Small-Molecule Inhibitors through Structure-Based Virtual Screening and Hit-Based Substructure Search

In this study, rapid structure-based virtual screening and hit-based substructure search were utilized to identify small molecules that disrupt the interaction of Keap1-Nrf2. Special emphasis was placed toward maximizing the exploration of chemical diversity of the initial hits while economically establishing informative structure-activity relationship (SAR) of novel scaffolds. Our most potent noncovalent inhibitor exhibits three times improved cellular activation in Nrf2 activation than the most active noncovalent Keap1 inhibitor known to date.

CXC and CC chemokines form mixed heterodimers: association free energies from molecular dynamics simulations and experimental correlations.

CXC and CC chemokines are involved in numerous biological processes, and their function in situ may be significantly influenced by heterodimer formation, as was recently reported, for example, for CXC chemokines CXCL4/PF4 and CXCL8/IL8 that interact to form heterodimers that modulate chemotactic and cell proliferation activities. Here we used molecular dynamics simulations to determine relative association free energies (overall average and per residue) for homo- and heterodimer pairs of CXC (CXCL4/PF4, CXCL8/IL8, CXCL1/Gro-α, and CXCL7/NAP-2) and CC (CCL5/RANTES, CCL2/MCP-1, and CCL8/MCP-2) chemokines. Even though structural homology among monomer folds of all CXC and CC chemokines permits heterodimer assembly, our calculated association free energies depend upon the particular pair of chemokines in terms of the net electrostatic and nonelectrostatic forces involved, as well as (for CC/CXC mixed chemokines) the selection of dimer type (CC or CXC). These relative free energies indicate that association of some pairs of chemokines is more favorable than others. Our approach is validated by correlation of calculated and experimentally determined free energies. Results are discussed in terms of CXC and CC chemokine function and have significant biological implications.

The surface constraint all atom model provides size independent results in calculations of hydration free energies

Consistent approaches for calculations of solvation free energies should provide results which are independent of the size of the simulation region. Simulations that use periodic boundary conditions and a standard Ewald treatment yield size dependent results. Corrections that can overcome this problem have been formulated, but have not yet been fully validated for solutes with general charge distributions. Furthermore, Ewald treatments of proteins may involve size dependent problems whose nature has not been explored by systematic studies. Here we demonstrate that our surface constraint all-atom solvent (SCAAS) model with its spherical boundary conditions (that include special polarization constraints) provides proper size independent results. It is also pointed out that this approach lends itself to an effective treatment of long-range interactions and offers a useful way of obtaining size independent free energies in studies of electrostatic effects in proteins.

A critical role for the loop region of the basic helix–loop–helix/leucine zipper protein Mlx in DNA binding and glucose-regulated transcription

The carbohydrate response element (ChoRE) is a cis-acting sequence found in the promoters of genes induced transcriptionally by glucose. The ChoRE is composed of two E box-like motifs that are separated by 5 bp and is recognized by two basic helix–loop–helix/leucine zipper (bHLH/LZ) proteins, ChREBP and Mlx, which heterodimerize to bind DNA. In this study, we demonstrate that two ChREBP/Mlx heterodimers interact to stabilize binding to the tandem E box-like motifs in the ChoRE. Based on a model structure that we generated of ChREBP/Mlx bound to the ChoRE, we hypothesized that intermolecular interactions between residues within the Mlx loop regions of adjacent heterodimers are responsible for stabilizing the complex. We tested this hypothesis by preparing Mlx variants in which the loop region was replaced with that of another family member or mutated at several key residues. These Mlx variants retained their ability to bind to a single perfect E-box motif as a heterodimer with ChREBP, but no longer bound to the ChoRE nor supported glucose responsive activity. In summary, our results support a model in which the loop regions of Mlx play an important functional role in mediating the coordinate binding of ChREBP/Mlx heterodimers to the ChoRE.

Triazole-Linked Inhibitors of Inosine Monophosphate Dehydrogenase from Human and Mycobacterium tuberculosis

The modular nature of nicotinamide adenine dinucleotide (NAD)-mimicking inosine monophsophate dehydrogenase (IMPDH) inhibitors has prompted us to investigate novel mycophenolic adenine dinucleotides (MAD) in which 1,2,3-triazole linkers were incorporated as isosteric replacements of the pyrophosphate linker. Synthesis and evaluation of these inhibitors led to identification of low nanomolar inhibitors of human IMPDH and more importantly the first potent inhibitor of IMPDH from Mycobacterium tuberculosis (mtIMPDH). Computational studies of these IMPDH enzymes helped rationalize the observed structure-activity relationships. Additionally, the first cloning, expression, purification and characterization of mtIMPDH is reported.