Monday, October 2, 2023

Disordered proteins: the next frontier in therapeutics?


Howard Stone at Princeton, Ozgur Sahin at Columbia, and their colleagues have written a wonderfully imaginative review of what they call “hydration solids” (Nature 619, 500; 2023 - here). By this I don’t mean to imply anything fantastical about it – I simply like this way of framing the notion that hydration water can play an important structural and mechanical role in some hygroscopic biological materials. The idea here is that the mechanical properties are governed by the hydration forces operative within a fluid-filled porous elastic medium. The notion is motivated and explored by studying the mechanical behaviour of bacterial spores, but the same principles might apply to wood, pollen grains, keratinous materials and silk. “Such ‘hydration solids’, which can exchange their essential constituent water with the environment and have it flow through the material, are potentially abundant in the environment”, they write.


There is a fascinating paper in PRL from Chunyi Zhang (Mike Klein’s group) at Temple University in Philadelphia that investigates why the dielectric permittivity of salt water can actually decrease as more salt is added (C. Zhang et al., Phys. Rev. Lett. 131, 076801; 2023 - here). Using a deep neural network trained on the results of density functional theory, the authors show that this is not some kind of saturation effect but arises because of the way the ionic hydration shells disrupt the hydrogen-bonded network of the water and thereby suppress the collective response to electric fields.


It has been recognized at least since the early 1970s (and explored by the late, great Jack Dunitz) that changes in enthalpy and in entropy of associations between biomolecules (such as receptor-ligand pairings), due for example to small changes in molecular structure, seem often to compensate for one another so as to entail little change in the Gibbs free energy of binding. Why this is so has been much debated. An intuitive explanation is that a more favourable enthalpic contribution to binding creates a corresponding decrease in conformational freedom and thus a loss in entropy. But consideration of that balance must also take into account changes in hydration due to reorganization of the local hydrogen-bonded network. The Whitesides group (Breiten et al., JACS  135, 15579; 2013) has argued that water reorganization is in fact the key source of enthalpy-entropy compensation. That idea is examined, and ultimately supported, in a study by Shensheng Chen and Zhen-Gang Wang at Caltech (J. Phys. Chem. B 127, 6825; 2023 - here), using MD simulations of model charged polymers. They find that the hydrophobic interactions resulting from reorganization of hydration water show temperature dependencies that can account for the close correlation between the deltaH and TdeltaS terms. The effects of electrostatic interactions and polymer conformational changes are, in comparison, minor. Water is, it seems, in control.

Liquid-liquid phase separation (LLPS) has become a vibrant topic in cell biology now that it’s clear cells make use of it in a variety of ways and circumstances for partitioning and sequestering biomolecules for purposes ranging from gene regulation to RNA splicing to stress responses. The globular droplets – condensates – formed in this process have a higher density than the surrounding cell fluid, but it’s still not really understood what characteristics of biomolecules promote this new phase. That understanding could be useful for being able to control the phase separation process for possible therapeutic purposes – or indeed for designing peptides to prevent pathogenic aggregation. The condensates are not biomolecular complexes in any real sense – the binding forces between the components seem to be rather weak and indiscriminate, and condensates typically contain proteins with some degree of disorder (intrinsically disordered proteins, IDPs), which tend to be promiscuous in their interactions. Debasis Saha and Biman Jana of the Indian Association for the Cultivation of Science in Kolkata have used MD simulations to investigate the factors that govern the interactions of model peptides, leading to dimerization, to try to get some handle on what is going on (J. Phys. Chem. B 127, 6656; 2023 - here). They consider the effect of charged residues such as arginine, as well as differing amounts of hydrophobicity in the chains, in altering the free energy surface for dimerization. For both positively and negatively charged peptides, the solvation water seems to play an important role, and is the dominant influence in the latter case. The upshot is that, if dimerization adequately reflects the condensate formation process, negatively charged peptides seem more likely to stay in the dilute phase.

Benjamin Schuler at Chicago and colleagues look at a similar issue: the interaction between the highly positively charged histone linker H1 and the highly negatively charged prothymosin α (ProTα) which acts as a “chaperone” that can help H1 disassociate from the histone (A. Chowdhury et al., PNAS 120, e2304036120; 2023 - here). Both are IDPs. They find that in this case there is a large entropic contribution to the binding coming from the release of counterions – a consideration that they expect to be most generally applicable to bio-polyelectrolyte interactions.  

Meanwhile, in a paper in bioRxiv [here], Saumyak Mukherjeee and Lars Schäfer at Bochum have looked at the thermodynamic driving forces that govern the formation of condensates from proteins with intrinsically disordered domains. Aggregation into the dense phase will involve changes in enthalpy and entropy due both to direct protein interactions and to changes in solvation. The authors conclude from MD simulations that in this case the most important factors are protein interaction enthalpy and the entropic effects of water release from the protein hydration shell into the bulk.

It seems likely that unravelling these factors governing IDP associations of various sorts will ultimately be valuable as we seek to make pharmaceutical interventions in molecular interactions of this kind that seem connected to disease (such as Alzheimer’s). Christine Lim at Cambridge and colleagues have developed a bioinformatics platform for identifying proteins involved in LLPS that seem to be potential therapeutic targets (C. M. Lim et al., PNAS 120, e2300215120; 2023 - here). They test it by looking at the in vitro phase behaviour of three targets that their scheme identifies. I don’t think it is too much to suggest that this points towards something of a new paradigm for therapeutics, in which the goal is not to develop some inhibitor that might compete with a protein’s normal ligand but rather to engineer collective and less selective interactions at a larger scale.

I’m intrigued by a paper in press in J. General Physiology (preprint here) by Alan Kay at the University of Iowa and Gerald Manning at Rutgers, arguing that what drives osmosis is still not fully understood and that the mechanism proposed by Peter Debye in 1923 is in fact the right one, despite being now largely forgotten. It is one thing to explain osmotic flow thermodynamically in terms of differences in chemical potential (the textbook account), but another to explain what actually drives the directional transport of water molecules. Manning and Kay say that diffusion alone is not able to account for the osmotic flux, which arises instead because of differential repulsive forces between the solute molecules and the two interfaces of the semipermeable membrane. This produces the equivalent of a hydrostatic pressure difference that drives the flux. I’m certainly not qualified to assess whether this revision of the textbook explanation is valid, but the history of the Debye model given in the paper is surely interesting in its own right.  

There is, admittedly, not much biology going on in the deep mantle of the Earth with temperatures of 1,000-2,000 K and pressures of up to 22 GPa. All the same, such conditions pose quite a test of molecular-dynamics water potential functions, which Roberto Car at Princeton, Giulia Galli at Chicago, and colleagues have put through their paces (C. Zhang et al., J. Phys. Chem B 127, 7011; 2023 - here). They set out to calculate the thermal conductivity of water in these conditions, using a potential found by fitting to density-functional theory using a deep-learning algorithm. The conductivity varies only slightly with temperature, decreasing from its ambient value, but is much more strongly (and positively) correlated with density. The heat transport of water at high P and T could have important implications for processes in the interiors of gas-giant planets.

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