Charge environments around phosphorylation sites in proteins

Background: Phosphorylation is a central feature in many biological processes. Structural analyses have identified the importance of charge-charge interactions, for example mediating phosphorylation-driven allosteric change and protein binding to phosphopeptides. Here, we examine computationally the prevalence of charge stabilisation around phosphorylated sites in the structural database, through comparison with locations that are not phosphorylated in the same structures. Results: A significant fraction of phosphorylated sites appear to be electrostatically stabilised, largely through interaction with sidechains. Some examples of stabilisation across a subunit interface are evident from calculations with biological units. When considering the immediately surrounding environment, in many cases favourable interactions are only apparent after conformational change that accompanies phosphorylation. A simple calculation of potential interactions at longer-range, applied to non-phosphorylated structures, recovers the separation exhibited by phosphorylated structures. In a study of sites in the Phospho.ELM dataset, for which structural annotation is provided by non-phosphorylated proteins, there is little separation of the known phospho-acceptor sites relative to background, even using the wider interaction radius. However, there are differences in the distributions of patch polarity for acceptor and background sites in the Phospho.ELM dataset. Conclusion: In this study, an easy to implement procedure is developed that could contribute to the identification of phospho-acceptor sites associated with charge-charge interactions and conformational change. Since the method gives information about potential anchoring interactions subsequent to phosphorylation, it could be combined with simulations that probe conformational change. Our analysis of the Phospho.ELM dataset also shows evidence for mediation of phosphorylation effects through (i) conformational change associated with making a solvent inaccessible phospho-acceptor site accessible, and (ii) modulation of protein-protein interactions

Simulation of non-specific protein–mRNA interactions

Protein–nucleic acid interactions exhibit varying degrees of specificity. Relatively high affinity, sequence-specific interactions, can be studied with structure determination, but lower affinity, non-specific interactions are also of biological importance. We report simulations that predict the population of nucleic acid paths around protein surfaces, and give binding constant differences for changes in the protein scaffold. The method is applied to the non-specific component of interactions between eIF4Es and messenger RNAs that are bound tightly at the cap site. Adding a fragment of eIF4G to the system changes both the population of mRNA paths and the protein–mRNA binding affinity, suggesting a potential role for non-specific interactions in modulating translational properties. Generally, the free energy simulation technique could work in harness with characterized tethering points to extend analysis of nucleic acid conformation, and its modulation by protein scaffolds

Mechanisms for stabilisation and the maintenance of solubility in proteins from thermophiles

BACKGROUND: The database of protein structures contains representatives from organisms with a range of growth temperatures. Various properties have been studied in a search for the molecular basis of protein adaptation to higher growth temperature. Charged groups have emerged as key distinguishing factors for proteins from thermophiles and mesophiles. RESULTS: A dataset of 291 thermophile-derived protein structures is compared with mesophile proteins. Calculations of electrostatic interactions support the importance of charges, but indicate that increases in charge contribution to folded state stabilisation do not generally correlate with the numbers of charged groups. Relative propensities of charged groups vary, such as the substitution of glutamic for aspartic acid sidechains. Calculations suggest an energetic basis, with less dehydration for longer sidechains. Most other properties studied show weak or insignificant separation of proteins from moderate thermophiles or hyperthermophiles and mesophiles, including an estimate of the difference in sidechain rotameric entropy upon protein folding. An exception is increased burial of alanine and proline residues and decreased burial of phenylalanine, methionine, tyrosine and tryptophan in hyperthermophile proteins compared to those from mesophiles. CONCLUSION: Since an increase in the number of charged groups for hyperthermophile proteins is separable from charged group contribution to folded state stability, we hypothesise that charged group propensity is important in the context of protein solubility and the prevention of aggregation. Accordingly we find some separation between mesophile and hyperthermophile proteins when looking at the largest surface patch that does not contain a charged sidechain. With regard to our observation that aromatic sidechains are less buried in hyperthermophile proteins, further analysis indicates that the placement of some of these groups may facilitate the reduction of folding fluctuations in proteins of the higher growth temperature organisms

Compuchtational prediction of expression and solubility of recombinant biopharmaceuticals

Protein based therapeutics have emerged as a successful class of pharmaceutical. However, it is well known that much of the current therapeutic protein discovery and development processes is based around existing molecular frameworks and that novel formats offer significant challenges for expression. Efficient production of protein is required to meet the growing demands and increasing expectation of the patients and healthcare providers. Major obstacles during biopharmaceutical production are linked to the efficiency of the protein expression system and the biophysical properties of proteinbased products that can lead to aggregation and subsequent problems for purification, quality and effectiveness. Computational tools have been developed to aid prediction of protein solubility and aggregation propensity to support enhanced certainty of optimal generation of product with desirable properties. In this study, we have used an in-house computational tool for prediction of soluble protein expression (developed around protein structure and surface electrostatic properties of human erythropoietin, HuEPO) was developed in E. coli and has been validated experimentally with several bacteriallyexpressed model protein variants. The application of the computational approach has been extended to mammalian expression platforms. A significant inverse correlation was observed between positive surface patches and the expressability of HuEPO in transient mammalian cells (HEK and CHO cell lines). Mechanistically the differential expression operates at a level post-transcriptionally, associated with ribosome-secretory complex engagement, protein stability or secretory processes. The results demonstrate the potential of application of a predictive computational algorithm as a design tool in rational protein engineering to improve expression of novel protein formats in mammalian systems as well as E.coli. In summary, optimization of molecular patches on the surface of proteins may be a viable strategy to enhance protein soluble expression and therefore a potential solution for development of novel proteins that might otherwise fit into the category of “difficult-toexpress” proteins

Thermodynamic Origin of Differential Excipient-Lysozyme Interactions

Understanding the intricate interplay of interactions between proteins, excipients, ions and water is important to achieve the effective purification and stable formulation of protein therapeutics. The free energy of lysozyme interacting with two kinds of polyanionic excipients, citrate and tripolyphosphate, together with sodium chloride and TRIS-buffer, are analysed in multiple-walker metadynamics simulations to understand why tripolyphosphate causes lysozyme to precipitate but citrate does not. The resulting multiscale decomposition of energy and entropy components for water, sodium chloride, excipients and lysozyme reveals that lysozyme is more stabilised by the interaction of tripolyphosphate with basic residues. This is accompanied by more sodium ions being released into solution from tripolyphosphate than for citrate, whilst the latter instead has more water molecules released into solution. Even though lysozyme aggregation is not directly probed in this study, these different mechanisms are suspected to drive the cross-linking between lysozyme molecules with vacant basic residues, ultimately leading to precipitation

Thermodynamic Origin of Differential Excipient-Lysozyme Interactions

From Frontiers via Jisc Publications RouterHistory: collection 2021, received 2021-03-31, accepted 2021-05-25, epub 2021-06-11Publication status: PublishedUnderstanding the intricate interplay of interactions between proteins, excipients, ions and water is important to achieve the effective purification and stable formulation of protein therapeutics. The free energy of lysozyme interacting with two kinds of polyanionic excipients, citrate and tripolyphosphate, together with sodium chloride and TRIS-buffer, are analysed in multiple-walker metadynamics simulations to understand why tripolyphosphate causes lysozyme to precipitate but citrate does not. The resulting multiscale decomposition of energy and entropy components for water, sodium chloride, excipients and lysozyme reveals that lysozyme is more stabilised by the interaction of tripolyphosphate with basic residues. This is accompanied by more sodium ions being released into solution from tripolyphosphate than for citrate, whilst the latter instead has more water molecules released into solution. Even though lysozyme aggregation is not directly probed in this study, these different mechanisms are suspected to drive the cross-linking between lysozyme molecules with vacant basic residues, ultimately leading to precipitation

PhosIDP: a web tool to visualize the location of phosphorylation sites in disordered regions

From Springer Nature via Jisc Publications RouterHistory: received 2021-03-15, accepted 2021-04-19, registration 2021-04-20, online 2021-05-11, pub-electronic 2021-05-11, collection 2021-12Publication status: PublishedFunder: Agency for Science, Technology and Research; doi: http://dx.doi.org/10.13039/501100001348; Grant(s): IDs H17/01/a0/010, IDs H17/01/a0/010Funder: Engineering and Physical Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000266; Grant(s): EP/N024796/1, EP/N024796/1Abstract: Charge is a key determinant of intrinsically disordered protein (IDP) and intrinsically disordered region (IDR) properties. IDPs and IDRs are enriched in sites of phosphorylation, which alters charge. Visualizing the degree to which phosphorylation modulates the charge profile of a sequence would assist in the functional interpretation of IDPs and IDRs. PhosIDP is a web tool that shows variation of charge and fold propensity upon phosphorylation. In combination with the displayed location of protein domains, the information provided by the web tool can lead to functional inferences for the consequences of phosphorylation. IDRs are components of many proteins that form biological condensates. It is shown that IDR charge, and its modulation by phosphorylation, is more tightly controlled for proteins that are essential for condensate formation than for those present in condensates but inessential

Visualization of poly(ADP-ribose) bound to PARG reveals inherent balance between exo- and endo-glycohydrolase activities

Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

From Springer Nature via Jisc Publications RouterHistory: received 2021-04-28, rev-recd 2021-06-02, accepted 2021-06-08, registration 2021-06-11, pub-electronic 2021-06-28, online 2021-06-28, pub-print 2021-07Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/P004237/1Abstract: Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.’s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1–15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems