ABSTRACT
The G4C2 hexanucleotide repeat expansion in the c9orf72 gene is a major genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), with the formation of G-quadruplexes directly linked to the development of these diseases. Cations play a crucial role in the formation and structure of G-quadruplexes. In this study, we investigated the impact of biologically relevant potassium ions on G-quadruplex structures and utilized 15N-labeled ammonium cations as a substitute for K+ ions to gain further insights into cation binding and exchange dynamics. Through nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we demonstrate that the single d(G4C2) repeat, in the presence of 15NH4+ ions, adopts a tetramolecular G-quadruplex with an all-syn quartet at the 5'-end. The movement of 15NH4+ ions through the central channel of the G-quadruplex, as well as to the bulk solution, is governed by the vacant cation binding site, in addition to the all-syn quartet at the 5'-end. Furthermore, the addition of K+ ions to G-quadruplexes folded in the presence of 15NH4+ ions induces stacking of G-quadruplexes via their 5'-end G-quartets, leading to the formation of stable higher-ordered species.
Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Frontotemporal Lobar Degeneration , G-Quadruplexes , Humans , Amyotrophic Lateral Sclerosis/genetics , Cations , PotassiumABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Chemometrics , Humans , Protein Stability , Antibodies, Monoclonal/chemistry , Protein Unfolding , Protein Conformation , Drug StabilityABSTRACT
Adenosine tripolyphosphate (ATP) is a small polyvalent anion that has recently been shown to interact with proteins and have a major impact on assembly processes involved in biomolecular condensate formation and protein aggregation. However, the nature of non-specific protein-ATP interactions and their effects on protein solubility are largely unknown. Here, the binding of ATP to the globular model protein is characterized in detail using X-ray crystallography and nuclear magnetic resonance (NMR). Using NMR, we identified six ATP binding sites on the lysozyme surface, with one known high-affinity nucleic acid binding site and five non-specific previously unknown sites with millimolar affinities that also bind tripolyphosphate (TPP). ATP binding occurs primarily through the polyphosphate moiety, which was confirmed by the X-ray structure of the lysozyme-ATP complex. Importantly, ATP binds preferentially to arginine over lysine in non-specific binding sites. ATP and TPP have similar effects on solution-phase protein-protein interactions. At low salt concentrations, ion binding to lysozyme causes precipitation, while at higher salt concentrations, redissolution occurs. The addition of an equimolar concentration of magnesium to ATP does not alter ATP binding affinities but prevents lysozyme precipitation. These findings have important implications for both protein crystallization and cell biology. Crystallization occurs readily in ATP solutions outside the well-established crystallization window. In the context of cell biology, the findings suggest that ATP binds non-specifically to folded proteins in physiological conditions. Based on the nature of the binding sites identified by NMR, we propose several mechanisms for how ATP binding can prevent the aggregation of natively folded proteins.
Subject(s)
Adenosine , Muramidase , Adenosine/metabolism , Muramidase/chemistry , Binding Sites , Polyphosphates , Adenosine Triphosphate/metabolism , Protein BindingABSTRACT
Surfactants are commonly used in biopharmaceutical formulations to stabilize proteins against aggregation. However, the choice of a suitable surfactant for a particular protein is decided mostly empirically, and their mechanism of action on molecular level is largely unknown. Here we show that a straightforward label-free method, saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, can be used to detect protein-surfactant interactions in formulations of a model protein, interferon alpha. We find that polysorbate 20 binds with its fatty acid to interferon, and that the binding is stronger at pH closer to the isoelectric point of the protein. In contrast, we did not detect interactions between poloxamer 407 and interferon alpha. Neither of the two surfactants affected the tertiary structure and the thermal stability of the protein as evident from circular dichroism and nanoDSF measurements. Interestingly, both surfactants inhibited the formation of subvisible particles during long-term storage, but only polysorbate 20 reduced the amount of small soluble aggregates detected by size-exclusion chromatography. This proof-of-principle study demonstrates how STD-NMR can be employed to quickly assess surfactant-protein interactions and support the choice of surfactant in protein formulation.
Subject(s)
Polysorbates , Surface-Active Agents , Surface-Active Agents/chemistry , Polysorbates/chemistry , Interferon-alpha , Magnetic Resonance Spectroscopy/methods , Proteins/chemistryABSTRACT
Photocaging is an attractive strategy to control molecular behaviour, for example, in chemical synthesis, interaction studies or photodynamic therapies. Here, we demonstrate that in situ illumination by the LED NMRtorch approach enables effective and controlled photocage release with simultaneous monitoring of subsequent reactions by solution NMR spectroscopy.
Subject(s)
Lighting , Delayed-Action Preparations , Magnetic Resonance SpectroscopyABSTRACT
RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Peptides/pharmacology , Protein Binding/drug effects , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/analysis , Signal Transduction/drug effects , Cell Line, Tumor , Cell Membrane/metabolism , Humans , Proto-Oncogene Proteins p21(ras)/metabolismABSTRACT
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.
Subject(s)
Anti-Infective Agents/chemistry , Drug Compounding/methods , Excipients/chemistry , High-Throughput Screening Assays/methods , Peptides/chemistry , Anti-Infective Agents/therapeutic use , Humans , Infections/drug therapy , Molecular Dynamics Simulation , Peptides/therapeutic use , Proton Magnetic Resonance Spectroscopy , Reproducibility of ResultsABSTRACT
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.
Subject(s)
Point Mutation/genetics , Pore Forming Cytotoxic Proteins/chemistry , Pore Forming Cytotoxic Proteins/genetics , Biophysics/methods , Calorimetry, Differential Scanning/methods , Dynamic Light Scattering/methods , Hydrogen-Ion Concentration , Peptides/chemistry , Peptides/genetics , Protein Aggregates/genetics , Protein Stability/drug effectsABSTRACT
One of the major challenges in formulation development of biopharmaceuticals is improving long-term storage stability, which is often achieved by addition of excipients to the final formulation. Finding the optimal excipient for a given protein is usually done using a trial-and-error approach, due to the lack of general understanding of how excipients work for a particular protein. Previously, preferential interactions (binding or exclusion) of excipients with proteins were postulated as a mechanism explaining diversity in the stabilisation effects. Weak preferential binding is however difficult to quantify experimentally, and the question remains whether the formulation process should seek excipients which preferentially bind with proteins, or not. Here, we apply solution NMR spectroscopy to comprehensively evaluate protein-excipient interactions between therapeutically relevant proteins and commonly used excipients. Additionally, we evaluate the effect of excipients on thermal and colloidal protein stability, on aggregation kinetics and protein storage stability at elevated temperatures. We show that there is a weak negative correlation between the strength of protein-excipient interactions and effect on enhancing protein thermal stability. We found that the overall protein-excipient binding per se can be a poor criterion for choosing excipients enhancing formulation stability. Experiments on a diverse set of excipients and test proteins reveal that while excipients affect all of the different aspects of protein stability, the effects are very much protein specific, and care must be taken to avoid apparent generalisations if a smaller dataset is being used.
Subject(s)
Biological Products/chemistry , Excipients/chemistry , Protein Binding/physiology , Proteins/chemistry , Chemistry, Pharmaceutical/methods , Drug Stability , Kinetics , Protein StabilityABSTRACT
The hexanucleotide expansion GGGGCC located in C9orf72 gene represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Since the discovery one of the non-exclusive mechanisms of expanded hexanucleotide G4C2 repeats involved in ALS and FTLD is RNA toxicity, which involves accumulation of pathological sense and antisense RNA transcripts. Formed RNA foci sequester RNA-binding proteins, causing their mislocalization and, thus, diminishing their biological function. Therefore, structures adopted by pathological RNA transcripts could have a key role in pathogenesis of ALS and FTLD. Utilizing NMR spectroscopy and complementary methods, we examined structures adopted by both guanine-rich sense and cytosine-rich antisense RNA oligonucleotides with four hexanucleotide repeats. While both oligonucleotides tend to form dimers and hairpins, the equilibrium of these structures differs with antisense oligonucleotide being more sensitive to changes in pH and sense oligonucleotide to temperature. In the presence of K+ ions, guanine-rich sense RNA oligonucleotide also adopts secondary structures called G-quadruplexes. Here, we also observed, for the first time, that antisense RNA oligonucleotide forms i-motifs under specific conditions. Moreover, simultaneous presence of sense and antisense RNA oligonucleotides promotes formation of heterodimer. Studied structural diversity of sense and antisense RNA transcripts not only further depicts the complex nature of neurodegenerative diseases but also reveals potential targets for drug design in treatment of ALS and FTLD.
Subject(s)
Nucleic Acid Conformation , Oligonucleotides, Antisense/chemistry , Oligonucleotides/chemistry , RNA, Antisense/chemistry , Repetitive Sequences, Nucleic Acid , Amyotrophic Lateral Sclerosis/etiology , Base Pairing , C9orf72 Protein/chemistry , C9orf72 Protein/genetics , Disease Susceptibility , Frontotemporal Dementia/etiology , Humans , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Oligonucleotides/genetics , Oligonucleotides, Antisense/genetics , RNA, Antisense/genetics , Spectrum Analysis , Structure-Activity Relationship , TemperatureABSTRACT
Understanding the effects of additives on therapeutic protein stability is of paramount importance for obtaining stable formulations. In this work, we apply several high- and medium-throughput methods to study the physical stability of a model monoclonal antibody at pH 5.0 and 6.5 in the presence of sucrose, arginine hydrochloride, and arginine glutamate. In low ionic strength buffer, the addition of salts reduces the antibody colloidal and thermal stability, attributed to screening of electrostatic interactions. The presence of glutamate ion in the arginine salt partially reduces the damaging effect of ionic strength increase. The addition of 280 mM sucrose shifts the thermal protein unfolding to a higher temperature. Arginine salts in the used concentration reduce the relative monomer yield after refolding from urea, whereas sucrose has a favorable effect on antibody refolding. In addition, we show 12-month long-term stability data and observe correlations between thermal protein stability, relative monomer yield after refolding, and monomer loss during storage. The monomer loss during storage is related to protein aggregation and formation of subvisible particles in some of the formulations. This study shows that the effect of commonly used additives on the long-term antibody physical stability can be predicted using orthogonal biophysical measurements.
Subject(s)
Antibodies, Monoclonal/chemistry , Arginine/chemistry , Dipeptides/chemistry , Sucrose/chemistry , Buffers , Colloids , Drug Compounding , Drug Stability , Drug Storage , High-Throughput Screening Assays , Hydrogen-Ion Concentration , Protein Aggregates , Protein Stability , Protein Unfolding , Temperature , Time FactorsABSTRACT
Peptide aptamers built using engineered scaffolds are a valuable alternative to monoclonal antibodies in many research applications because of their smaller size, versatility, specificity for chosen targets, and ease of production. However, inserting peptides needed for target binding may affect the aptamer structure, in turn compromising its activity. We have shown previously that a stefin A-based protein scaffold with AU1 and Myc peptide insertions (SQT-1C) spontaneously forms dimers and tetramers and that inserted loops mediate this process. In the present study, we show that SQT-1C forms tetramers by self-association of dimers and determine the kinetics of monomer-dimer and dimer-tetramer transitions. Using site-directed mutagenesis, we show that while slow domain swapping defines the rate of dimerization, conserved proline P80 is involved in the tetramerization process. We also demonstrate that the addition of a disulphide bond at the base of the engineered loop prevents domain swapping and dimer formation, also preventing subsequent tetramerization. Formation of SQT-1C oligomers compromises the presentation of inserted peptides for target molecule binding, diminishing aptamer activity; however, the introduction of the disulphide bond locking the monomeric state enables maximum specific aptamer activity, while also increasing its thermal and colloidal stability. We conclude that stabilizing scaffold proteins by adding disulphide bonds at peptide insertion sites might be a useful approach in preventing binding-epitope-driven oligomerization, enabling creation of very stable aptamers with maximum binding activity.
ABSTRACT
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.
Subject(s)
Cystatins/chemistry , Protein Engineering , Crystallography, X-Ray , Epitopes/chemistry , Polymerization , Protein ConformationABSTRACT
Colloidal stability is among the key challenges the pharmaceutical industry faces during the production and manufacturing of protein therapeutics. Self-association and aggregation processes can not only impair therapeutic efficacy but also induce immunogenic responses in patients. Aggregation-prone regions (APRs) consisting of hydrophobic patches are commonly identified as the source for colloidal instability, and rational strategies to mitigate aggregation propensity often require genetic engineering to eliminate hydrophobic amino acid residues. Here, we investigate cucurbit[7]uril (CB[7]), a water-soluble macrocycle able to form host-guest complexes with aromatic amino acid residues, as a potential excipient to mitigate protein aggregation propensity. Two monoclonal antibodies (mAbs), one harboring an APR and one lacking an APR, were first assessed for their colloidal stability (measured as the translational diffusion coefficient) in the presence and absence of CB[7] using dynamic light scattering. Due to the presence of a tryptophan residue within the APR, we were able to monitor changes in intrinsic fluorescence in response to increasing concentrations of CB[7]. Isothermal titration calorimetry and NMR spectroscopy were then used to characterize the putative host-guest interaction. Our results suggest a stabilizing effect of CB[7] on the aggregation-prone mAb, due to the specific interaction of CB[7] with aromatic amino acid residues located within the APR. This provides a starting point for exploring CB[7] as a candidate excipient for the formulation of aggregation-prone mAbs.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Excipients/chemistry , Excipients/metabolism , Macrocyclic Compounds/chemistry , Macrocyclic Compounds/metabolism , Amino Acids/metabolism , Binding Sites, Antibody , Calorimetry , Colloids/chemistry , Drug Compounding , Drug Stability , Dynamic Light Scattering , Hydrophobic and Hydrophilic Interactions , Magnetic Resonance Spectroscopy , Protein Binding , Solubility , Water/chemistryABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/chemistry , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Protein Conformation , Protein StabilityABSTRACT
The G4C2 hexanucleotide repeat expansion mutation (HREM) in C9ORF72, represents the most common mutation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Three main disease mechanisms have been proposed to date: C9ORF72 haploinsufficiency, RNA toxicity, and accumulation of dipeptide repeat proteins. Pure GC content of the HREM potentially enables the formation of various non-B DNA structures such as G-quadruplexes and i-motifs. These structures are proposed to act as promoters and regulatory elements affecting replication, transcription and translation of the surrounding region. G-quadruplexes have already been shown on the G-rich sense DNA and RNA strands (G4C2)n, the structure of the anti-sense (G2C4)n strand remains unresolved. Similar C-rich sequences may, under acidic conditions, form i-motifs consisting of two parallel duplexes in a head to tail orientation held together by hemi-protonated C(+)-C pairs. We show that d(G2C4)n repeats do form i-motif and protonated hairpins even under near-physiological conditions. Rather than forming a DNA duplex, i-motifs persist even in the presence of the sense strand. This preferential formation of G-quadruplex and i-motif/hairpin structures over duplex DNA, may explain HREM replicational and transcriptional instability. Furthermore, i-motifs/hairpins can represent a novel pharmacological target for C9ORF72 associated ALS and FTLD.
Subject(s)
DNA Repeat Expansion/genetics , Proteins/chemistry , Proteins/genetics , Base Composition , C9orf72 Protein , Circular Dichroism , DNA, Antisense , Humans , Magnetic Resonance Spectroscopy , Nucleic Acid Conformation , Nucleotide MotifsABSTRACT
The G4C2 hexanucleotide repeat expansion, located in the first intron of the C9ORF72 gene, represents a major genetic hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several hypotheses have been proposed on how the transcribed repeat RNA leads to the development of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. However, despite their importance, factors affecting the transcription of expanded-repeat RNA are not well known. As transcription is dependent on the DNA containing the expanded repeats, it is crucial to understand its structure. G-quadruplexes are known to affect expression on the level of DNA, therefore whether they form on the expanded-repeat DNA constitutes an important biological question. Using nuclear magnetic resonance and circular dichroism spectroscopy we show that DNA G4C2 with varying number of repeats d(G4C2)n form planar guanine quartets characteristic of G-quadruplexes. Additionally, we show DNA G-quadruplexes can form inter- and intra-molecularly in either parallel or anti-parallel orientation, based on d(G4C2) sequence length. This potential structural heterogeneity of longer disease-relevant repeats should therefore be taken into account when studying their role in disease pathogenesis.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , DNA Repeat Expansion/genetics , Frontotemporal Lobar Degeneration/genetics , G-Quadruplexes , Proteins/genetics , C9orf72 Protein , Circular Dichroism , DNA/genetics , Humans , Introns/genetics , Magnetic Resonance Spectroscopy , RNA/genetics , Transcription, GeneticABSTRACT
Integral equation approaches, based on the Ornstein-Zernike equation, provide a fast way to calculate phase diagrams and thermodynamic properties of systems as opposed to time-consuming and computationally expensive computer simulations. However, when employing integral equations it is necessary to introduce simplifications. The Ornstein-Zernike equation merely relates two unknown functions h(r) and c(r), and another relation (closer) between these two functions is needed. The later function cannot be obtained in a closed form and it is always in some approximations. Various approximations exist with each of its own advantages and disadvantages. In this work we extensively tested hyper-netted chain, Percus-Yevick, Kovalenko-Hirata, and Rogers-Young closure on an interaction model with core-softened potential. Convergence domain was established for each method. We calculated pair distribution functions, pressure, and excess energy. Results were compared with Monte Carlo simulation results and literature data from molecular dynamics simulations.