In-silico study of the interactions between acylated glucagon like-peptide-1 analogues and the native receptor.

We have performed a series of multiple molecular dynamics (MD) simulations of glucagon-like peptide-1 (GLP-1) and acylated GLP-1 analogues in complex with the endogenous receptor (GLP-1R) to obtain a molecular understanding of how fatty acid (FA) chain structure, acylation position on the peptide, and presence of a linker affect the binding. MD simulations were analysed to extract heatmaps of receptor-peptide interaction patterns and to determine the free energy of binding using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach. The extracted free energies from MM-PBSA calculations are in qualitative agreement with experimentally determined potencies. Furthermore, the interaction patterns seen in the receptor-GLP-1 complex simulations resemble previously reported binding interactions validating the simulations. Analysing the receptor-GLP-1 analogue complex simulations, we found that the major differences between the systems stem from FA interactions and positioning of acylation in the peptide. Hydrophobic interactions between the FA chain and a hydrophobic patch on the extracellular domain contribute significantly to the binding affinity. Acylation on Lys26 resulted in noticeably more interactions between the FA chain and the extracellular domain hydrophobic patch than found for acylation on Lys34 and Lys38, respectively. The presence of a charged linker between the peptide and FA chain can potentially stabilise the complex by forming hydrogen bonds to arginine residues in the linker region between the extracellular domain and the transmembrane domain. A molecular understanding of the fatty acid structure and its effect on binding provides important insights into designing acylated agonists for GLP-1R.Communicated by Ramaswamy H. Sarma.

pH- and concentration-dependent supramolecular assembly of a fungal defensin plectasin variant into helical non-amyloid fibrils.

Self-assembly and fibril formation play important roles in protein behaviour. Amyloid fibril formation is well-studied due to its role in neurodegenerative diseases and characterized by refolding of the protein into predominantly ß-sheet form. However, much less is known about the assembly of proteins into other types of supramolecular structures. Using cryo-electron microscopy at a resolution of 1.97 Å, we show that a triple-mutant of the anti-microbial peptide plectasin, PPI42, assembles into helical non-amyloid fibrils. The in vitro anti-microbial activity was determined and shown to be enhanced compared to the wildtype. Plectasin contains a cysteine-stabilised α-helix-ß-sheet structure, which remains intact upon fibril formation. Two protofilaments form a right-handed protein fibril. The fibril formation is reversible and follows sigmoidal kinetics with a pH- and concentration dependent equilibrium between soluble monomer and protein fibril. This high-resolution structure reveals that α/ß proteins can natively assemble into fibrils.

Development of a fast screening method for selecting excipients in formulations using MD simulations, NMR and microscale thermophoresis.

Development of peptide therapeutics generally involves screening of excipients that inhibit peptide-peptide interactions, hence aggregation, and improve peptide stability. We used the therapeutic peptide plectasin to develop a fast screening method that combines microscale thermophoresis titration assays and molecular dynamics simulations to relatively rank the excipients with respect to binding affinity and to study key peptide-excipient interaction hotspots on a molecular level, respectively. Additionally, 1H-13C-HSQC NMR titration experiments were performed to validate the fast screening approach. The NMR results are in qualitative agreement with results from the fast screening method demonstrating that this approach can be reliably applied to other peptides and proteins as a fast screening method to relatively rank excipients and predict possible excipient binding sites.

The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening.

Therapeutic peptides and proteins show enormous potential in the pharmaceutical market, but high costs in discovery and development are limiting factors so far. Single or multiple point mutations are commonly introduced in protein drugs to increase their binding affinity or selectivity. They can also induce adverse properties, which might be overlooked in a functional screen, such as a decreased colloidal or thermal stability, leading to problems in later stages of the development. In this study, we address the effect of point mutations on the stability of the 4.4 kDa antimicrobial peptide plectasin, as a case study. We combined a systematic high-throughput biophysical screen of the peptide thermal and colloidal stability using dynamic light scattering and differential scanning calorimetry with structure-based methods including small-angle X-ray scattering, analytical ultracentrifugation, and nuclear magnetic resonance spectroscopy. Additionally, we applied molecular dynamics simulations to link obtained protein stability parameters to the protein's molecular structure. Despite their predicted structural similarities, all four plectasin variants showed substantially different behavior in solution. We observed an increasing propensity of plectasin to aggregate at a higher pH, and the introduced mutations influenced the type of aggregation. Our strategy for systematically assessing the stability and aggregation of protein drugs is generally applicable and is of particular relevance, given the increasing number of protein drugs in development.

Albumin-neprilysin fusion protein: understanding stability using small angle X-ray scattering and molecular dynamic simulations.

Fusion technology is widely used in protein-drug development to increase activity, stability, and bioavailability of protein therapeutics. Fusion proteins, like any other type of biopharmaceuticals, need to remain stable during production and storage. Due to the high complexity and additional intramolecular interactions, it is not possible to predict the behavior of fusion proteins based on the behavior the individual proteins. Therefore, understanding the stability of fusion proteins on the molecular level is crucial for the development of biopharmaceuticals. The current study on the albumin-neprilysin (HSA-NEP) fusion protein uses a combination of thermal and chemical unfolding with small angle X-ray scattering and molecular dynamics simulations to show a correlation between decreasing stability and increasing repulsive interactions, which is unusual for most biopharmaceuticals. It is also seen that HSA-NEP is not fully flexible: it is present in both compact and extended conformations. Additionally, the volume fraction of each conformation changes with pH. Finally, the presence of NaCl and arginine increases stability at pH 6.5, but decreases stability at pH 5.0.

Studies of the oligomerisation mechanism of a cystatin-based engineered protein scaffold.

Engineered protein scaffolds are an alternative to monoclonal antibodies in research and drug design due to their small size, ease of production, versatility, and specificity for chosen targets. One key consideration when engineering such proteins is retaining the original scaffold structure and stability upon insertion of target-binding loops. SQT is a stefin A derived scaffold protein that was used as a model to study possible problems associated with solution behaviour of such aptamers. We used an SQT variant with AU1 and Myc insertion peptides (SQT-1C) to study the effect of peptide insertions on protein structure and oligomerisation. The X-ray structure of monomeric SQT-1C revealed a cystatin-like fold. Furthermore, we show that SQT-1C readily forms dimers and tetramers in solution. NMR revealed that these oligomers are symmetrical, with inserted loops comprising the interaction interface. Two possible mechanisms of oligomerisation are compared using molecular dynamics simulations, with domain swap oligomerisation being thermodynamically favoured. We show that retained secondary structure upon peptide insertion is not indicative of unaltered 3D structure and solution behaviour. Therefore, additional methods should be employed to comprehensively assess the consequences of peptide insertions in all aptamers, particularly as uncharacterized oligomerisation may alter binding epitope presentation and affect functional efficiency.

Conformational Stability Study of a Therapeutic Peptide Plectasin Using Molecular Dynamics Simulations in Combination with NMR.

Plectasin is a small, cysteine-rich peptide antibiotic which belongs to the class of antimicrobial peptides and has potential antibacterial activity against various Gram-positive bacteria. In the current study, the effect of pH and ionic strength (NaCl) on the conformational stability of plectasin variants has been investigated. At all physiochemical conditions, peptide secondary structures are intact throughout simulations. However, flexibility increases with pH because of the change in electrostatics around the distinct anionic tetrapeptide (9-12) stretch. Furthermore, plectasin interactions with NaCl were measured by determining the preferential interaction coefficients, Γ23. Generally, wild-type plectasin has higher preference for sodium ions as 9ASP is mutated in other variants. Overall, the Γ23 trend with pH for the two salt conditions remain the same for all variants predominately having accumulation of sodium ions around 10GLU and 12ASP. Insignificant changes in the overall peptide conformational stability are in agreement with the fact that plectasin has three cystines. Thermodynamic integration molecular dynamics simulations supplemented with nuclear magnetic resonance were employed to determine the degree of involvement of three different cystines to the overall structural integrity of the peptide. Both methods show the same order of cystine reduction and complete unfolding is observed only upon reduction of all cystines.