Reza Salari

The Native GCN4 Leucine-Zipper Domain Does Not Uniquely Specify a Dimeric Oligomerization State

The dimerization domain of the yeast transcription factor GCN4, one of the first coiled-coil proteins to be structurally characterized at high resolution, has served as the basis for numerous fundamental studies on α-helical folding. Mutations in the GCN4 leucine zipper are known to change its preferred oligomerization state from dimeric to trimeric or tetrameric; however, the wild-type sequence has been assumed to encode a two-chain assembly exclusively. Here we demonstrate that the GCN4 coiled-coil domain can populate either a dimer or trimer fold, depending on environment. We report high-resolution crystal structures of the wild-type sequence in dimeric and trimeric assemblies. Biophysical measurements suggest populations of both oligomerization states under certain experimental conditions in solution. We use parallel tempering molecular dynamics simulations on the microsecond time scale to compare the stability of the dimer and trimer folded states in isolation. In total, our results suggest that the folding behavior of the well-studied GCN4 leucine-zipper domain is more complex than was previously appreciated. Our results have implications in ongoing efforts to establish predictive algorithms for coiled-coil folds and the selection of coiled-coil model systems for design and mutational studies where oligomerization state specificity is an important consideration.

Computational investigation of cholesterol binding sites on mitochondrial VDAC.

The mitochondrial voltage-dependent anion channel (VDAC) allows passage of ions and metabolites across the mitochondrial outer membrane. Cholesterol binds mammalian VDAC, and we investigated the effects of binding to human VDAC1 with atomistic molecular dynamics simulations that totaled 1.4 μs. We docked cholesterol to specific sites on VDAC that were previously identified with NMR, and we tested the reliability of multiple docking results in each site with simulations. The most favorable binding modes were used to build a VDAC model with cholesterol occupying five unique sites, and during multiple 100 ns simulations, cholesterol stably and reproducibly remained bound to the protein. For comparison, VDAC was simulated in systems with identical components but with cholesterol initially unbound. The dynamics of loops that connect adjacent β-strands were most affected by bound cholesterol, with the averaged root-mean-square fluctuation (RMSF) of multiple residues altered by 20–30%. Cholesterol binding also stabilized charged residues inside the channel and localized the surrounding electrostatic potentials. Despite this, ion diffusion through the channel was not significantly affected by bound cholesterol, as evidenced by multi-ion potential of mean force measurements. Although we observed modest effects of cholesterol on the open channel, our model will be particularly useful in experiments that investigate how cholesterol affects VDAC function under applied electrochemical forces and also how other ligands and proteins interact with the channel.

Effects of high temperature on desolvation costs of salt bridges across protein binding interfaces: similarities and differences between implicit and explicit solvent models.

The role of salt bridges in protein-protein binding is largely determined by the costs of desolvating the oppositely charged members of the salt bridge upon binding. On the basis of Poisson-Boltzmann (PB) implicit solvent calculations, it has been proposed that the reduced desolvation penalties of salt bridges at high temperatures provide one explanation for the increased abundance of salt bridges in hyperthermophilic proteins. Here, for the first time, we directly compare the PB implicit solvent model with several explicit water models in computing the effects of extremely high temperature (i.e., 100 °C) on the desolvation penalties of salt bridges across protein-protein interfaces. With the exception of two outliers, the desolvation costs at 100 °C from implicit and explicit solvent calculations are of similar magnitudes and significantly reduced relative to 25 °C. The two outliers correspond to salt bridges that are both buried and part of a salt bridge network, a challenging case that should be considered in the development of fast solvation models.

Direct Observations of Shifts in the β-Sheet Register of a Protein-Peptide Complex Using Explicit Solvent Simulations

Using explicit solvent molecular dynamics simulations, we were able to obtain direct observations of shifts in the hydrogen-bonding register of an intermolecular β-sheet protein-peptide complex. The β-sheet is formed between the FHA domain of cancer marker protein Ki67 (Ki67FHA) and a peptide fragment of the hNIFK signaling protein. Potential encounter complexes of the Ki67FHA receptor and hNIFK peptide are misregistered states of the β-sheet. Rearrangements of one of these misregistered states to the native state were captured in three independent simulations. All three rearrangements occurred by a common mechanism: an aromatic residue of the peptide (F263) anchors into a transient hydrophobic pocket of the receptor to facilitate the formation of native hydrogen bonds. To our knowledge, these simulations provide the first atomically detailed visualizations of a mechanism by which nature might correct for errors in the alignment of intermolecular β-sheets.

A streamlined, general approach for computing ligand binding free energies and its application to GPCR-bound cholesterol

The theory of receptor-ligand binding equilibria has long been well-established in biochemistry, and was primarily constructed to describe dilute aqueous solutions. Accordingly, few computational approaches have been developed for making quantitative predictions of binding probabilities in environments other than dilute isotropic solution. Existing techniques, ranging from simple automated docking procedures to sophisticated thermodynamics-based methods, have been developed with soluble proteins in mind. Biologically and pharmacologically relevant protein-ligand interactions often occur in complex environments, including lamellar phases like membranes and crowded, nondilute solutions. Here, we revisit the theoretical bases of ligand binding equilibria, avoiding overly specific assumptions that are nearly always made when describing receptor-ligand binding. Building on this formalism, we extend the asymptotically exact Alchemical Free Energy Perturbation technique to quantifying occupancies of sites on proteins in a complex bulk, including phase-separated, anisotropic, or nondilute solutions, using a thermodynamically consistent and easily generalized approach that resolves several ambiguities of current frameworks. To incorporate the complex bulk without overcomplicating the overall thermodynamic cycle, we simplify the common approach for ligand restraints by using a single distance-from-bound-configuration (DBC) ligand restraint during AFEP decoupling from protein. DBC restraints should be generalizable to binding modes of most small molecules, even those with strong orientational dependence. We apply this approach to compute the likelihood that membrane cholesterol binds to known crystallographic sites on three GPCRs (β2-adrenergic, 5HT-2B, and μ-opioid) at a range of concentrations. Nonideality of cholesterol in a binary cholesterol:phosphatidylcholine (POPC) bilayer is characterized and consistently incorporated into the interpretation. We find that the three sites exhibit very different affinities for cholesterol: The site on the adrenergic receptor is predicted to be high affinity, with 50% occupancy for 1:109 CHOL:POPC mixtures. The sites on the 5HT-2B and μ-opioid receptor are predicted to be lower affinity, with 50% occupancy for 1:103 CHOL:POPC and 1:102 CHOL:POPC, respectively. These results could not have been predicted from the crystal structures alone.

Absolute Affinity Calculations for Cholesterol Binding to G-Protein Coupled Receptors (GPCR)

Cholesterol is an essential constituent of most eukaryotic plasma membranes, and is critical for native function of many membrane proteins, including pentameric ligand gated ion channels (pLGICs) and G-protein coupled receptors (GPCRs). GPCRs are a family of integral membrane proteins that play a critical role in signal transduction across the membrane and are estimated to be the target for more than a third of all drugs. Although the number of available crystal structures for GPCRs in complex with various agonists, antagonists, and G proteins has expanded rapidly, the physiological relevance of direct and indirect interactions of GPCRs with cholesterol and other lipids in mixed membranes is poorly understood. For instance, resolved cholesterol is a common feature of most GPCR crystal structures, indicating a potentially significant structural role. The ubiquity of the resolved cholesterol in GPCR structures, even across GPCRs with a range of cholesterol sensitivities, largely precludes interpretation of the directly bound cholesterol molecule in the context of known functional effects of cholesterol. In the simplest mechanism, differential affinities of cholesterol for the cholesterol binding site on a range of GPCRs could predict differential sensitivities, but testing such a mechanism is complicated by numerous challenges in experimental measurements of lipid binding affinity. We recently developed a framework for calculating absolute binding affinity of lipophilic ligands for transmembrane proteins, which we used to predict cholesterol occupation ratio of intersubunit cavities of GluCl, a eukaryotic pLGIC, at physiological membrane concentrations of cholesterol. Here we employ this approach to calculate absolute cholesterol binding free energies and estimate average binding site occupancies for a range of GPCRs.