Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation.

Growth hormone (GH) acts via JAK2 and LYN to regulate growth, metabolism, and neural function. However, the relationship between these tyrosine kinases remains enigmatic. Through an interdisciplinary approach combining cell biology, structural biology, computation, and single-particle tracking on live cells, we find overlapping LYN and JAK2 Box1-Box2-binding regions in GH receptor (GHR). Our data implicate direct competition between JAK2 and LYN for GHR binding and imply divergent signaling profiles. We show that GHR exhibits distinct mobility states within the cell membrane and that activation of LYN by GH mediates GHR immobilization, thereby initiating its nanoclustering in the membrane. Importantly, we observe that LYN mediates cytokine receptor degradation, thereby controlling receptor turnover and activity, and this applies to related cytokine receptors. Our study offers insight into the molecular interactions of LYN with GHR and highlights important functions for LYN in regulating GHR nanoclustering, signaling, and degradation, traits broadly relevant to many cytokine receptors.

Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding.

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Dynamics of Human Serum Transferrin in Varying Physicochemical Conditions Explored by Using Molecular Dynamics Simulations.

Conformational stability of human serum transferrin (Tf) at varying pH values and salt and excipient concentrations were investigated using molecular dynamics (MD) simulations, and the results are compared with previously published small-angle X-ray scattering (SAXS) experiments. SAXS study showed that at pH 5, Tf is predominantly present in a partially open (PO) form, and the factions of PO differ based on the physicochemical condition and drift toward the closed form (HO) as the pH increases. Tf is a bilobal glycoprotein that is composed of homologous halves termed the N- and C-lobes. The current study shows that the protonation of Y188 and K206 at pH 5 is the primary conformational drive into PO, which shifts toward the closed (HO) conformer as the pH increases. Furthermore, at pH 6.5, PO is unfavorable due to negative charge-charge repulsion at the N/C-lobe interface linker region causing increased hinge distance when compared to HO, which has favorable attractive electrostatic interactions in this region. Subsequently, the effect of salt concentration was studied at 70 and 140 mM NaCl. At 70 mM NaCl and pH 5, chloride ions bind strongly in the N-lobe iron-binding site, whereas these interactions are weak at pH 6.5. With increasing salt concentration at pH 5, the regions surrounding the N-lobe iron-binding site are saturated, and as a consequence, sodium and chloride ions accumulate into the bulk. Additionally, protein-excipient interactions were investigated. At pH 5, the excipients interact in similar loop regions, E89-T93, and D416-D420, located in the N- and C-lobes of the HO conformer, respectively. It is anticipated that interactions of additives in these two loop regions cause conformational changes that lead to iron-coordinating residues in the N-lobe to drift away from iron and thus drive HO to PO conversion. Furthermore, at pH 6.5 and 140 mM histidine, these interactions are negligible leading to the stabilization of HO.

DSDBASE 2.0: updated version of DiSulphide dataBASE, a database on disulphide bonds in proteins.

Disulphide bonds are stabilizing crosslinks in proteins and serve to enhance their thermal stability. In proteins that are small and rich in disulphide bonds, they could be the major determining factor for the choice of conformational state since their constraints on appropriate backbone conformation can be substantial. Such crosslinks and their positional conservation could itself enable protein family and functional association. Despite the importance of the field, there is no comprehensive database on disulphide crosslinks that is available to the public. Herein we provide information on disulphides in DSDBASE2.0, an updated and significantly expanded database that is freely available, fully annotated and manually curated database on native and modelled disulphides. The web interface also provides several useful computational tools that have been specifically developed for proteins containing disulphide crosslinks. The modelling of disulphide crosslinks is performed using stereochemical criteria, coded within our Modelling of Disulphides in Proteins (MODIP) algorithm. The inclusion of modelled disulphides potentially enhances the loop database substantially, thereby permitting the recognition of compatible polypeptide segments that could serve as templates for immediate modelling. The DSDBASE2.0 database has been updated to include 153,944 PDB entries, 216,096 native and 20,153,850 modelled disulphide bond segments from PDB January 2021 release. The current database also provides a resource to user-friendly search for multiple disulphide bond containing loops, along with annotation of their function using GO and subcellular localization of the query. Furthermore, it is possible to obtain the three-dimensional models of disulphide-rich small proteins using an independent algorithm, RANMOD, that generates and examines random, but allowed backbone conformations of the polypeptide. DSDBASE2.0 still remains the largest open-access repository that organizes all disulphide bonds of proteins on a single platform. The database can be accessed from http://caps.ncbs.res.in/dsdbase2.

Electrostatics Drive Oligomerization and Aggregation of Human Interferon Alpha-2a.

Aggregation is a common phenomenon in the field of protein therapeutics and can lead to function loss or immunogenic patient responses. Two strategies are currently used to reduce aggregation: (1) finding a suitable formulation, which is labor-intensive and requires large protein quantities, or (2) engineering the protein, which requires extensive knowledge about the protein aggregation pathway. We present a biophysical characterization of the oligomerization and aggregation processes by Interferon alpha-2a (IFNα-2a), a protein drug with antiviral and immunomodulatory properties. This study combines experimental high throughput screening with detailed investigations by small-angle X-ray scattering and analytical ultracentrifugation. Metropolis Monte Carlo simulations are used to gain insight into the underlying intermolecular interactions. IFNα-2a forms soluble oligomers that are controlled by a fast pH and concentration-dependent equilibrium. Close to the isoelectric point of 6, IFNα-2a forms insoluble aggregates which can be prevented by adding salt. We show that monomer attraction is driven mainly by molecular anisotropic dipole-dipole interactions that increase with increasing pH. Repulsion is due to monopole-monopole interactions and depends on the charge of IFNα-2a. The study highlights how combining multiple methods helps to systematically dissect the molecular mechanisms driving oligomer formation and to design ultimately efficient strategies for preventing detrimental protein aggregation.

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.

Small angle X-ray scattering and molecular dynamic simulations provide molecular insight for stability of recombinant human transferrin.

Transferrin is an attractive candidate for drug delivery due to its ability to cross the blood brain barrier. However, in order to be able to use it for therapeutic purposes, it is important to investigate how its stability depends on different formulation conditions. Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes. Lowering pH induces opening of the transferrin N-lobe, which results in a negative effect on the stability. Presence of NaCl or arginine at low pH enhances the opening and has a negative impact on the overall protein stability. STATEMENT OF SIGNIFICANCE: Protein-based therapeutics have become an essential part of medical treatment. They are highly specific, have high affinity and fewer off-target effects. However, stabilization of proteins is critical, time-consuming, and expensive, and it is not yet possible to predict the behavior of proteins under different conditions. The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and therefore with high potential in drug delivery. Combination of high throughput unfolding techniques and structural studies, using small angle X-ray scattering and molecular dynamic simulations, allows us to understand the behavior of transferrin on a molecular level.

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.

Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization.

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

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.