Tuesday, January 17, 2017

Hydration water in drug design

Electrostatic interactions with lipid heads groups retard water molecules near the surface of a membrane. But how are those dynamics affected by a membrane protein? Lars Schäfer at the Ruhr University of Bochum and colleagues attempt to answer that question using (ODNP-enhanced) NMR and simulations to deduce water motions (O. Fisette et al., JACS 138, 11526; 2016 – paper here). They conclude that the water-protein interactions have a weaker retarding effect, and dominate only at distances of more than 10 Å above the membrane surface. Moreover, the protein (here annexin B12) and membrane effects are additive. This creates a gradient in water entropy with distance from the surface, with potential consequences for recognition and binding events involving membrane proteins.

The hydration environment of membrane protein annexin B12 in a lipid membrane.

A technique called oriented-sample solid-state NMR can supply information about the water-accessibility of individual residues of membrane proteins in situ, say Gianluigi Veglia and colleagues at the University of Minnesota (A. Dicke et al., JPCB 120, 10959; 2016 – paper here). They’ve used the method to gather this information for the archetypal small transmembrane protein sarcolipin in synthetic bilayers, and find that, as one might expect, there is a relatively smooth gradient of water accessibility with increasing depth within the membrane.

Does the denaturing effect of osmolytes such as urea and guanidinium chloride depend on concentration? Experiments using FRET and SAXS have produced conflicting results, and Robert Best at the NIH and colleagues try to resolve the matter using simulations (W. Zheng et al., JACS 138, 11702; 2016 – paper here). Their test case is the intrinsically disordered protein ACTR, for which they calculate the chain swelling and radius of gyration as a function of denaturant concentration. The protein does indeed swell to a degree proportional to concentration, but the researchers show that nevertheless the structural changes are consistent with SAXS results that appear to show no change in radius of gyration. The denaturant effects operate by a direct mechanism of weak association with the protein.

What is there is a mixture of denaturants such as urea and stabilizing osmolytes such as TMAO? It seems that TMAO can counteract urea’s effects, but how exactly is the hydrophobic interaction affected in that environment? Indrajit Tah and Jagannath Mondal at the Tata Institute in Hyderabad look into that question via simulations of a model hydrophobic polymer and polystyrene (JPCB 120, 10969; 2016 – paper here). In contrast to what is observed with proteins, for the hydrophobic polymer TMAO actually reinforces the destabilizing effect of urea. This seems to be due to the different direct interactions with the polymer chain: for proteins, TMAO is excluded from the surface while urea remains bound, whereas for the hydrophobic polymer both osmolytes may individually bind to the surface. In this latter case, exclusion of TMAO by urea in the mixed solution depletes the opportunities of TMAO to stabilize the collapsed state.

Guangzhao Zhang and colleagues at the South China University of Technology in Guangzhou look at the same denaturant-inhibiting effect of betaine, this time with lysozyme as the model protein (J. Chen et al., JPCB 120, 12327; 2016 – paper here). Using proton NMR, they conclude that in this case betaine interacts directly with urea to form dimers, removing the urea from the protein surface where otherwise it interacts directly to stabilize the denatured state.

Ion-specific “Hofmeister” effects on protein stability and aggregation are still not fully understood at the molecular level. Simon Ebbinghaus at the Ruhr University of Bochum and colleagues seek insights through thermodynamic (DSC) measurements on bovine ribonuclease A (M. Senske et al., PCCP 18, 29698; 2016 – paper here). By measuring ion effects over the whole temperature- and concentration-dependent landscape of protein stability, they find a very complicated picture, due to a complex interplay of contributions. At low concentrations, electrostatic (non-ion-specific) effects dominate, but at higher concentrations there is ion specificity. It’s hard (for me, anyway) to summarize the findings, but I believe it if fair to say that the authors are seeking a unified molecular picture that helps to explain not only ion effects but also those of non-electrolyte cosolutes on protein stability, in terms of a balance between entropic and enthalpic contributions to the excess free energy.

A belated addition from the Bochum group: Yao Xu and Martina Havenith have provided a nice summary of recent work using THz spectroscopy to look at ps-scale collective hydrogen-bond dynamics in the hydration shells of proteins, and the role that they play in molecular-recognition processes (JCP 143, 10.1063/1.4934504; 2015 – paper here).

A further use of THz spectroscopy to investigate hydration is reported by Y Ogawa and colleagues ay Kyoto University (K. Shiraga et al., Appl. Phys. Lett. 106, 253701; 2016 – paper here). Their aim is simply to get some bulk estimate of how much of the water in a cell (they use HeLa cells) has retarded dynamics – a question explored here in the context of the now more or less obsolete notion of “biological water” in cells. Their answer: about a quarter of the total water content has reorientational dynamics slower than the bulk, presumably because of its involvement in biomolecular hydration. This is more than the 10-15% reported previously in prokaryotes and human red blood cells. But of course the absolute numbers must depend on where one places the thresholds in a dynamical continuum.

The voltage-dependent proton transport channel Hv1 is implicated in diseases ranging from cancer to some forms of brain damage. This makes it a potential drug target, and some inhibitors seem to expel bound water from the pore when they bind. Because this water forms intermittent hydrogen-bonded clusters and water wires, it seems likely that there’s a hydration-related entropic contribution to the binding free energy. With this in mind, Mike Klein at Temple University and colleagues have used modeling and simulations to look at water fluctuations in the pore, so as to identify potential binding sites (E. Gianti et al., PNAS 113, E8359 2016 – paper here). Their analysis reveals two such sites: one the binding site known already, another at the outlet of the proton pathway, both of them associated with maximal fluctuation, apparently on the brink of a drying transition, and in locations where replacement by a hydrophobic ligand is optimal. The researchers say that the second, new site should therefore also be considered as a locus of drug design.

The same group has also considered the mechanism of proton transport (S C. van Keulen et al., JPCB 10.1021/acs.jpcb.6b08339; 2016 – paper here). It’s been suggested previously that this occurs via Grotthuss hopping along a water wire. But the results of these quantum/molecular mechanics simulations suggest that instead the proton hops between three acidic residues via mediating water molecules.

How a proton (circled in purple) makes its way down the channel of Hv1: via a series of water-mediated hops between acidic residues.

More proton transport: on the basis of FTIR experiments, Udita Brahmachari and Bridgette Barry at Georgia Tech say that a crucial stage of the oxygen-forming S-cycle of photosynthesis involves the insertion of a proton into a hydrogen-bonded water network (JPCB 120, 11464; 2016 – paper here). Thus bound water here acts as a catalytic proton acceptor and donor.

The catalytic water network in the S3-S0 stage of the photosynthetic cycle.

Jonathan Nickells at Oak Ridge and coworkers have characterized the general hydration environment of green fluorescent protein using neutron scattering spectroscopy to probe the dynamics (S. Perticaroli et al., JACS 10.1021/jacs.6b08845; 2016 – paper here). These dynamics are slowed over just two hydration shells: by a factor of 4-10 in the first shell and 2-5 in the second.

The hydration of GFP.

In comparison, Keiichiro Shiraga and colleagues at Kyoto see dynamical perturbations out to three or four hydration layers (a distance of around 8.5 Å) around albumin, based on THz spectroscopy (K. Shiraga et al., Biophys. J. 111, 2629; 2016 – paper here). They say that the hydrogen-bond network in the hydration layers seems to be less defective than that in the bulk, even though there seems to be greater distortion of the network away from tetrahedral.

Meanwhile, Monique Tourell and Konstantin Momot at the Queensland University of Technology have zeroed in on the single-water-molecule bridges that link parts of the peptide chains in collagen (JPCB 120, 12432; 2016 – paper here). Some experimental studies have apparently implied that the waters are “ice-like” in their dynamics, although I’m not clear quite what this is meant to imply. In any event, these MD simulations suggest otherwise: the waters exhibit strongly anisotropic rotation in which a single molecule might flip back and forth many times while remaining resident at the bridging site for more than 100 picoseconds.

When a particle is solvated at the air-water surface, fluctuations of the interface such as capillary waves may contribute to the solvation free energy – the solute might dampen the fluctuations, for example. Kaustubh Rane and Nico van der Vegt set out to quantify this using Monte Carlo simulations (JPCB 120, 9697; 2016 – paper here). They find that the contribution of fluctuations is not negligible in general, and that the dampening effect is a generic one that doesn’t depend on the chemical nature of solute or solvent. However, the strength of the interactions between ions and water will determine the magnitude of the effect of fluctuations, so that one can expect ion-specific propensities towards proximity to the water surface.

Ariel Fernández, now at the Argentine Mathematics Intitute, has been for some time exploring the role of hydrogen-bond frustration in protein-protein and protein-ligand interactions. The idea here is that such frustration contributes to interfacial tension via its effects on “non-Debye” polarization. Now he has studied how “minimal frustration” might guide these molecular assembly processes and be exploited in drug design, potentially enabling a high degree of binding selectivity (FEBS Lett. 590, 3481; 2016 – paper here).

There’s more on the use of hydration information for drug design from Gerhard Klebe of the University of Marburg and colleagues (S. G. Krimmer et al., J. Med. Chem. 59, 10530; 2016 – paper here). They say that optimizing the water layers covering ligands bound to their target – here hydrophobic inhibitors of thermolysin – can boost the enthalpic contribution to binding free energy. MD simulations enabled the prediction of high binding affinity for a series of ligands, one of which then proved to have 50 times better binding affinity than the known (and patented) parent ligand. This is a really nice piece of work, showing that it’s not just trapped or displaced water molecules that are important in drug design but also the final bound-ligand hydration profile.

The hydration structure of the best drug candidate for binding in the hydrophobic pocket of thermolysin.

Nested fullerenes, or “carbon onions”, cluster in water. Adam Makarucha have used MD simulations to look at the size- and shape-dependence of the effect (A. J. Kakarucha et al., JPCB 120, 11018; 2016 – paper here). As one might expect for a hydrophobic surface, there is layering of water at the surface of these particles, and the disruption of the hydrogen-bond network increases with increasing particle size because of the increased shape anisotropy: the tendency for the larger fullerene shells to become faceted with vertices (where the pentagonal rings sit).

To what extent are the bulk properties of pure water altered by confinement? Debates about confinement-induced changes in “water structure” have sometimes tended to overlook generic effects such as layering of a liquid close to a wall. Roland Netz and colleagues at the Free University of Berlin bring some clarity to the problem by using MS simulations to look at changes in the dielectric properties of water due to confinement-induced correlations in the polarization of neighbouring water molecules (A. Schlaich et al., Phys. Rev. Lett. 117, 048001; 2016 – paper here). They find that these effects – specifically an anticorrelation for neighbouing molecules – result in a significantly decreased dielectric response perpendicular (but not so much parallel) to a pair of walls (here consisting of closely packed decanol monolayers) separated by up to a nanometre or so. This behaviour has obvious consequences for, say, water’s ability to screen electrostatic interactions between closely spaced surfaces (of proteins or lipid membranes, say). [There’s also an APS Physics comment piece on this here]

The existence of a quasiliquid layer on the surface of ice below 273 K is now fairly well attested. Mischa Bonn and Ellen Backus of the MPI for Polymer Research at Mainz and their colleagues have studied this layer using SFG and simulations (M. A. Sánchez et al., PNAS 114, 227; 2016 – paper here). They find evidence of a stepwise transition from a single to a double bilayer of water molecules around 257 K. They say that there is evidence for the single bilayer being quasiliquid all the way down to 235 K.