Carmay Lim

Metal binding affinity and selectivity in metalloproteins: insights from computational studies.

This review highlights insights gained from computational studies on protein-metal recognition. We systematically dissect the various factors governing metal binding affinity and selectivity in proteins starting from (a) the intrinsic properties of the metal and neighboring metal cations (if present), to (b) the primary coordination sphere, (c) the second coordination shell, (d) the protein matrix, (e) the bulk solvent, and (f) competing non-protein ligands from the surrounding biological environment. The results herein reveal the fundamental principles and the molecular bases underlying protein-metal recognition, which serve as a guide to engineer novel metalloproteins with programmed properties.

Factors Governing the Na+ vs K+ Selectivity in Sodium Ion Channels

Monovalent Na(+) and K(+) ion channels, specialized pore-forming proteins that play crucial biological roles such as controlling cardiac, skeletal, and smooth muscle contraction, are characterized by a remarkable metal selectivity, conducting the native cation while rejecting its monovalent contender and other ions present in the cellular/extracellular milieu. Compared to K(+) channels, the principles governing Na(+) vs K(+) selectivity in both epithelial and voltage-gated Na(+) channels are much less well understood due mainly to the lack of high-resolution 3D structures. Thus, many questions remain. It is not clear if the serines lining the pore of epithelial Na(+) channel bind to the metal cation via their backbone or side chain O atoms and why substituting the Lys lining the pore of voltage-gated Na(+) channels to another residue such as Arg drastically reduces or even reverses the Na(+)/K(+) selectivity. This work systematically evaluates the effects of various factors such as (i) the number, chemical type, and charge of the pores coordinating groups, (ii) the hydration number and coordination number of the metal cation, and (iii) the solvent exposure and the size/rigidity of the pore on the Na(+) vs K(+) selectivity in model Na(+) channel selectivity filters (the narrowest part of the pore) using a combined density functional theory/continuum dielectric approach. The results reveal that the Na(+) channels selectivity for Na(+) over K(+) increases if (1) the pore provides three rather than four protein ligands to coordinate to the metal ion, (2) the protein ligands have strong charge-donating ability such as Asp/Glu carboxylate or backbone carbonyl groups, (3) the passing Na(+) is bare or less well hydrated inside the filter than the competing K(+), and (4) the pore is relatively rigid, constricted, and solvent exposed. They also reveal that factors favoring Na(+)/K(+) selectivity in Na(+) channels generally disfavor K(+)/Na(+) selectivity in K(+) channels and vice versa. The different selectivity principles for the K(+) and Na(+) channels are consistent with the different architecture, composition, and properties of their selectivity filters. They provide clues to the metal-binding site structure in the selectivity filters of epithelial and voltage-gated Na(+) channels.

A simple biophysical model emulates budding yeast chromosome condensation

Mitotic chromosomes were one of the first cell biological structures to be described, yet their molecular architecture remains poorly understood. We have devised a simple biophysical model of a 300 kb-long nucleosome chain, the size of a budding yeast chromosome, constrained by interactions between binding sites of the chromosomal condensin complex, a key component of interphase and mitotic chromosomes. Comparisons of computational and experimental (4C) interaction maps, and other biophysical features, allow us to predict a mode of condensin action. Stochastic condensin-mediated pairwise interactions along the nucleosome chain generate native-like chromosome features and recapitulate chromosome compaction and individualization during mitotic condensation. Higher order interactions between condensin binding sites explain the data less well. Our results suggest that basic assumptions about chromatin behavior go a long way to explain chromosome architecture and are able to generate a molecular model of what the inside of a chromosome is likely to look like. DOI: http://dx.doi.org/10.7554/eLife.05565.001

DR_bind: a web server for predicting DNA-binding residues from the protein structure based on electrostatics, evolution and geometry

DR_bind is a web server that automatically predicts DNA-binding residues, given the respective protein structure based on (i) electrostatics, (ii) evolution and (iii) geometry. In contrast to machine-learning methods, DR_bind does not require a training data set or any parameters. It predicts DNA-binding residues by detecting a cluster of conserved, solvent-accessible residues that are electrostatically stabilized upon mutation to Asp−/Glu−. The server requires as input the DNA-binding protein structure in PDB format and outputs a downloadable text file of the predicted DNA-binding residues, a 3D visualization of the predicted residues highlighted in the given protein structure, and a downloadable PyMol script for visualization of the results. Calibration on 83 and 55 non-redundant DNA-bound and DNA-free protein structures yielded a DNA-binding residue prediction accuracy/precision of 90/47% and 88/42%, respectively. Since DR_bind does not require any training using protein–DNA complex structures, it may predict DNA-binding residues in novel structures of DNA-binding proteins resulting from structural genomics projects with no conservation data. The DR_bind server is freely available with no login requirement at http://dnasite.limlab.ibms.sinica.edu.tw.

Common physical basis of macromolecule-binding sites in proteins

Protein–DNA/RNA/protein interactions play critical roles in many biological functions. Previous studies have focused on the different features characterizing the different macromolecule-binding sites and approaches to detect these sites. However, no common unique signature of these sites had been reported. Thus, this work aims to provide a ‘common’ principle dictating the location of the different macromolecule-binding sites founded upon fundamental principles of binding thermodynamics. To achieve this aim, a comprehensive set of structurally nonhomologous DNA-, RNA-, obligate protein- and nonobligate protein-binding proteins, both free and bound to their respective macromolecules, was created and a novel strategy for detecting clusters of residues with electrostatic or steric strain given the protein structure was developed. The results show that regardless of the macromolecule type, the binding strength and conformational changes upon binding, macromolecule-binding sites are energetically less stable than nonmacromolecule-binding sites. They also reveal new energetic features distinguishing DNA- from RNA-binding sites and obligate protein- from nonobligate protein-binding sites in both free/bound protein structures.

A structural-alphabet-based strategy for finding structural motifs across protein families.

Proteins with insignificant sequence and overall structure similarity may still share locally conserved contiguous structural segments; i.e. structural/3D motifs. Most methods for finding 3D motifs require a known motif to search for other similar structures or functionally/structurally crucial residues. Here, without requiring a query motif or essential residues, a fully automated method for discovering 3D motifs of various sizes across protein families with different folds based on a 16-letter structural alphabet is presented. It was applied to structurally non-redundant proteins bound to DNA, RNA, obligate/non-obligate proteins as well as free DNA-binding proteins (DBPs) and proteins with known structures but unknown function. Its usefulness was illustrated by analyzing the 3D motifs found in DBPs. A non-specific motif was found with a ‘corner’ architecture that confers a stable scaffold and enables diverse interactions, making it suitable for binding not only DNA but also RNA and proteins. Furthermore, DNA-specific motifs present ‘only’ in DBPs were discovered. The motifs found can provide useful guidelines in detecting binding sites and computational protein redesign.

Ion Selectivity Strategies of Sodium Channel Selectivity Filters

CONSPECTUS: Sodium ion channels selectively transport Na(+) cations across the cell membrane. These integral parts of the cell machinery are implicated in regulating the cardiac, skeletal and smooth muscle contraction, nerve impulses, salt and water homeostasis, as well as pain and taste perception. Their malfunction often results in various channelopathies of the heart, brain, skeletal muscles, and lung; thus, sodium channels are key drug targets for various disorders including cardiac arrhythmias, heart attack, stroke, migraine, epilepsy, pain, cancer, and autoimmune disorders. The ability of sodium channels to discriminate the native Na(+) among other competing ions in the surrounding fluids is crucial for proper cellular functions. The selectivity filter (SF), the narrowest part of the channels open pore, lined with amino acid residues that specifically interact with the permeating ion, plays a major role in determining Na(+) selectivity. Different sodium channels have different SFs, which vary in the symmetry, number, charge, arrangement, and chemical type of the metal-ligating groups and pore size: epithelial/degenerin/acid-sensing ion channels have generally trimeric SFs lined with three conserved neutral serines and/or backbone carbonyls; eukaryotic sodium channels have EKEE, EEKE, DKEA, and DEKA SFs with an invariant positively charged lysine from the second or third domain; and bacterial voltage-gated sodium (Nav) channels exhibit symmetrical EEEE SFs, reminiscent of eukaryotic voltage-gated calcium channels. How do these different sodium channel SFs achieve high selectivity for Na(+) over its key rivals, K(+) and Ca(2+)? What factors govern the metal competition in these SFs and which of these factors are exploited to achieve Na(+) selectivity in the different sodium channel SFs? The free energies for replacing K(+) or Ca(2+) bound inside different model SFs with Na(+), evaluated by a combination of density functional theory and continuum dielectric calculations, have shed light on these questions. The SFs of epithelial and eukaryotic Nav channels select Na(+) by providing an optimal number and ligating strength of metal ligands as well as a rigid pore whose size fits the cognate Na(+) ideally. On the other hand, the SFs of bacterial Nav channels select Na(+), as the protein matrix attenuates ion-protein interactions relative to ion-solvent interactions by enlarging the pore and allowing water to enter, so the ion interacts indirectly with the conserved glutamates via bridging water molecules. This shows how these various SFs have adapted to the specific physicochemical properties of the native ion, using different strategies to select Na(+) among its contenders.

Evolution of Eukaryotic Ion Channels: Principles Underlying the Conversion of Ca2+-Selective to Na+-Selective Channels

Ion selectivity of four-domain voltage-gated Ca(2+) and Na(+) channels, which is controlled by the selectivity filter (the narrowest region of an open pore), is crucial for electrical signaling. Over billions of years of evolution, mutation of the Glu from domain II/III in the EEEE/DEEA selectivity filters of Ca(2+)-selective channels to Lys made these channels Na(+)-selective. Why Lys is sufficient for Na(+) selectivity and why the DKEA selectivity filter is less Na(+)-selective than the DEKA one are intriguing, fundamental questions. By computing the free energy for replacing Ca(2+) inside model selectivity filters with Na(+), we find that the nonmetal-ligating Lys in the DKEA/DEKA selectivity filter attenuates metal-protein interactions to such an extent that solvation effects become dominant, favoring Na(+). It constricts and rigidifies the DEKA pore to bind Na(+) optimally, highlighting the importance of lysines nonobvious structural role, in addition to its electrostatic role, in the selectivity of Na(+) over Ca(2+).

Solvation free energies of polar molecular solutes: Application of the two-sphere Born radius in continuum models of solvation

A two-sphere description of the effective Born radius for spherical ions was found in previous work to yield accurate free energies for spherical ions. This effective Born radius (Reff) was identified as the mean of the ionic radius (Rion) and the distance to the first peak of the ion–oxygen/hydrogen radial charge or number density distribution function (Rgmax); i.e., Reff=(Rion+Rgmax)/2. To see whether this prescription also applies to the solvation of nonspherical polar molecules, it was used in finite-difference Poisson methods as well as in Kirkwood and generalized Born models to compute solvation free energies of model diatomic molecules of varying interatomic bond distances. Hydration free energies for the same model systems were also derived from free energy simulations in the presence of explicit water molecules. The good agreement between explicit solvent results and continuum solvent results with the two-sphere Born radius indicates that the latter description provides the required solute–solven...

Two potential therapeutic antibodies bind to a peptide segment of membrane-bound IgE in different conformations

IgE mediates hypersensitivity reactions responsible for most allergic diseases, which affect 20-40% of the population in developed countries. A 52-residue domain of membrane-bound IgE (mIgE) called CεmX is currently a target for developing therapeutic antibodies; however, its structure is unknown. Here we show that two antibodies with therapeutic potential in IgE-mediated allergic diseases, which can cause cytolytic effects on mIgE-expressing B lymphocytes and downregulate IgE production, target different conformations of an intrinsically disordered region (IDR) in the extracellular CεmX domain. We provide an important example of antibodies targeting an extracellular IDR of a receptor on the surface of intended target cells. We also provide fundamental structural characteristics unique to human mIgE, which may stimulate further studies to investigate whether other monoclonal antibodies (mAbs) targeting intrinsically disordered peptide segments or vaccine-like products targeting IDRs of a membrane protein can be developed.