ABSTRACT
In this investigation, we tested the hypothesis that a physiologically based pharmacokinetic (PBPK) model incorporating measured in vitro metrics of off-target binding can largely explain the inter-antibody variability in monoclonal antibody (mAb) pharmacokinetics (PK). A diverse panel of 83 mAbs was evaluated for PK in wild-type mice and subjected to 10 in vitro assays to measure major physiochemical attributes. After excluding for target-mediated elimination and immunogenicity, 56 of the remaining mAbs with an eight-fold variability in the area under the curve (AUC0-672h: 1.74 × 106 -1.38 × 107 ngâh/mL) and 10-fold difference in clearance (2.55-26.4 mL/day/kg) formed the training set for this investigation. Using a PBPK framework, mAb-dependent coefficients F1 and F2 modulating pinocytosis rate and convective transport, respectively, were estimated for each mAb with mostly good precision (coefficient of variation (CV%) <30%). F1 was estimated to be the mean and standard deviation of 0.961 ± 0.593, and F2 was estimated to be 2.13 ± 2.62. Using principal component analysis to correlate the regressed values of F1/F2 versus the multidimensional dataset composed of our panel of in vitro assays, we found that heparin chromatography retention time emerged as the predictive covariate to the mAb-specific F1, whereas F2 variability cannot be well explained by these assays. A sigmoidal relationship between F1 and the identified covariate was incorporated within the PBPK framework. A sensitivity analysis suggested plasma concentrations to be most sensitive to F1 when F1 > 1. The predictive utility of the developed PBPK model was evaluated against a separate panel of 14 mAbs biased toward high clearance, among which area under the curve of PK data of 12 mAbs was predicted within 2.5-fold error, and the positive and negative predictive values for clearance prediction were 85% and 100%, respectively. MAb heparin chromatography assay output allowed a priori identification of mAb candidates with unfavorable PK.
Subject(s)
Antibodies, Monoclonal , Models, Biological , Mice , Animals , Pinocytosis , Biological Assay , HeparinABSTRACT
Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.
Subject(s)
Antibodies, Monoclonal , Drug Discovery , Animals , Mice , Antibodies, Monoclonal/chemistry , Computer Simulation , Recombinant Proteins , ViscosityABSTRACT
INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Yshaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartmentspecific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for diseaseassociated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cellbased assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiledcoil hub that tethers two separate mRNPremodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoanspecific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an Nterminal Sshaped αhelical solenoid followed by a coiledcoil oligomerization element, numerous Raninteracting domains, an E3 ligase domain, and a Cterminal prolylisomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their Nterminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cellbased assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryoET density matched the dimensions of the CFNC coiledcoil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiledcoil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryoET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our nearatomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].
Subject(s)
Cytoplasm , Fungal Proteins , Nuclear Pore Complex Proteins , Nuclear Pore , RNA Transport , RNA, Messenger , Chaetomium , Cryoelectron Microscopy , Cytoplasm/chemistry , Fungal Proteins/chemistry , Humans , Molecular Chaperones/chemistry , Nuclear Pore/chemistry , Nuclear Pore Complex Proteins/chemistry , Protein Conformation , RNA, Messenger/metabolismABSTRACT
Frataxin is a highly conserved mitochondrial protein whose deficiency in humans results in Friedreich's ataxia (FRDA), an autosomal recessive disorder characterized by progressive ataxia and cardiomyopathy. Although its cellular function is still not fully clear, the fact that frataxin plays a crucial role in Fe-S assembly on the scaffold protein Isu is well accepted. In the present paper, we report the characterization of eight frataxin variants having alterations on two putative functional regions: the alpha1/beta1 acidic ridge and the conserved beta-sheet surface. We report that frataxin iron-binding capacity is quite robust: even when five of the most conserved residues from the putative iron-binding region are altered, at least two iron atoms per monomer can be bound, although with decreased affinity. Furthermore, we conclude that the acidic ridge is designed to favour function over stability. The negative charges have a functional role, but at the same time significantly impair frataxin's stability. Removing five of those charges results in a thermal stabilization of approximately 24 degrees C and reduces the inherent conformational plasticity. Alterations on the conserved beta-sheet residues have only a modest impact on the protein stability, highlighting the functional importance of residues 122-124.
Subject(s)
Iron-Binding Proteins/chemistry , Iron-Binding Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Friedreich Ataxia/genetics , Friedreich Ataxia/metabolism , Humans , Iron/metabolism , Iron-Binding Proteins/genetics , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Stability , Protein Structure, Secondary , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , FrataxinABSTRACT
The worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented and the impact on public health and the global economy continues to be devastating. Although early therapies such as prophylactic antibodies and vaccines show great promise, there are concerns about the long-term efficacy and universal applicability of these therapies as the virus continues to mutate. Thus, protein-based immunogens that can quickly respond to viral changes remain of continued interest. The Spike protein, the main immunogen of this virus, displays a highly dynamic trimeric structure that presents a challenge for therapeutic development. Here, guided by the structure of the Spike trimer, we rationally design new Spike constructs that show a uniquely high stability profile while simultaneously remaining locked into the immunogen-desirable prefusion state. Furthermore, our approach emphasizes the relationship between the highly conserved S2 region and structurally dynamic Receptor Binding Domains (RBD) to enable vaccine development as well as the generation of antibodies able to resist viral mutation.
Subject(s)
Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites/genetics , Binding Sites/immunology , COVID-19/immunology , COVID-19/pathology , Cell Line , HEK293 Cells , Humans , Protein Domains/genetics , Protein Domains/immunology , Protein Stability , SARS-CoV-2/geneticsABSTRACT
Bispecific antibodies, engineered to recognize two targets simultaneously, demonstrate exceptional clinical potential for the therapeutic intervention of complex diseases. However, these molecules are often composed of multiple polypeptide chains of differing sequences. To meet industrial scale productivity, enforcing the correct quaternary assembly of these chains is critical. Here, we describe Chain Selectivity Assessment (CSA), a high-throughput method to rationally select parental monoclonal antibodies (mAbs) to make bispecific antibodies requiring correct heavy/light chain pairing. By deploying CSA, we have successfully identified mAbs that exhibit a native preference toward cognate chain pairing that enables the production of hetero-IgGs without additional engineering. Furthermore, CSA also identified rare light chains (LCs) that permit positive binding of the non-cognate arm in the common LC hetero-IgGs, also without engineering. This rational selection of parental mAbs with favorable developability characteristics is critical to the successful development of bispecific molecules with optimal manufacturability properties.
Subject(s)
Antibodies, Bispecific/immunology , Antibodies, Monoclonal/immunology , Immunoglobulin G/immunology , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/immunology , Antibody Affinity/immunology , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Chromatography, Liquid/methods , Electrophoresis, Capillary/methods , Electrophoresis, Polyacrylamide Gel/methods , HEK293 Cells , Humans , Mass Spectrometry/methods , Protein Engineering/methodsABSTRACT
Frataxin is a mitochondrial protein that is defective in Friedreich's ataxia resulting in iron accumulation and an environment prone to Fenton reactions. We report that frataxin is susceptible to carbonylation and nitration modifications in residues from the beta-sheet surface (Tyr143, Tyr174, Tyr205 and Trp155). Frataxin functions are not significantly affected: frataxin-mediated protection against ROS is still observed, as well as iron-binding (5 Fe(3+)mol(-1), K(d) from 13-36 microM) necessary for the metallochaperone activity. However, the protein is up to 1.0 kcal mol(-1) destabilized, with conformational opening. Interestingly, the strictly conserved Trp155, which is mutated in patients, may be a functional hotspot in frataxin.
Subject(s)
Iron-Binding Proteins/chemistry , Oxidative Stress , Protein Carbonylation , Tryptophan/chemistry , Amino Acid Sequence , Conserved Sequence , Humans , Iron-Binding Proteins/metabolism , Nitrites/chemistry , Protein Stability , Protein Structure, Secondary , Tryptophan/metabolism , FrataxinABSTRACT
Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble/aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin.
Subject(s)
Friedreich Ataxia/genetics , Iron-Binding Proteins/chemistry , Iron-Binding Proteins/genetics , Point Mutation , Humans , Iron/metabolism , Iron-Binding Proteins/metabolism , Kinetics , Models, Molecular , Protein Conformation , Protein Folding , Thermodynamics , Trypsin/metabolism , FrataxinABSTRACT
The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1â¢Nxt1 from mRNAs. Here, we report crystal structures of Gle1â¢Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP6, unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1â¢Nup42â¢DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.
Subject(s)
Nuclear Pore/chemistry , Nuclear Pore/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Active Transport, Cell Nucleus , Chaetomium/genetics , Chaetomium/metabolism , DEAD-box RNA Helicases/chemistry , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Models, Biological , Models, Molecular , Nuclear Pore/genetics , Nuclear Pore Complex Proteins/chemistry , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Phytic Acid/metabolism , RNA Transport , RNA, Messenger/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Determining macromolecular structures from X-ray data with resolution worse than 3â Å remains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0â Å resolution F1-ATPase data set and a 4.5â Å resolution SecYEG-SecA complex data set. All of the models were automatically built by the method to Rfree values of between 28.9 and 39.9% and were free from the initial model bias.
ABSTRACT
The neurodegenerative disorder FRDA (Friedreich's ataxia) results from a deficiency in frataxin, a putative iron chaperone, and is due to the presence of a high number of GAA repeats in the coding regions of both alleles of the frataxin gene, which impair protein expression. However, some FRDA patients are heterozygous for this triplet expansion and contain a deleterious point mutation on the other allele. In the present study, we investigated whether two particular FRDA-associated frataxin mutants, I154F and W155R, result in unfolded protein as a consequence of a severe structural modification. A detailed comparison of the conformational properties of the wild-type and mutant proteins combining biophysical and biochemical methodologies was undertaken. We show that the FRDA mutants retain the native fold under physiological conditions, but are differentially destabilized as reflected both by their reduced thermodynamic stability and a higher tendency towards proteolytic digestion. The I154F mutant has the strongest effect on fold stability as expected from the fact that the mutated residue contributes to the hydrophobic core formation. Functionally, the iron-binding properties of the mutant frataxins are found to be partly impaired. The apparently paradoxical situation of having clinically aggressive frataxin variants which are folded and are only significantly less stable than the wild-type form in a given adverse physiological stress condition is discussed and contextualized in terms of a mechanism determining the pathology of FRDA heterozygous.
Subject(s)
Friedreich Ataxia/metabolism , Iron-Binding Proteins/genetics , Iron-Binding Proteins/metabolism , Enzyme Stability , Humans , Iron-Binding Proteins/chemistry , Models, Molecular , Mutation , Protein Conformation , Protein Folding , Thermodynamics , FrataxinABSTRACT
The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here we present the crystal structure of a reconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central "triskelion" of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ~16 megadalton of its mass.
Subject(s)
Nuclear Pore Complex Proteins/chemistry , Nuclear Pore/ultrastructure , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/ultrastructure , Crystallography, X-Ray , Humans , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
Numerous human diseases are caused by protein folding defects where the protein may become more susceptible to degradation or aggregation. Aberrant protein folding can affect the kinetic stability of the proteins even if these proteins appear to be soluble in vivo. Experimental discrimination between functional properly folded and misfolded nonfunctional conformers is not always straightforward at near physiological conditions. The differences in the kinetic behavior of two initially folded frataxin clinical variants were examined using a high affinity chaperonin kinetic trap approach at 25 °C. The kinetically stable wild type frataxin (FXN) shows no visible partitioning onto the chaperonin. In contrast, the clinical variants FXN-p.Asp122Tyr and FXN-p.Ile154Phe kinetically populate partial folded forms that tightly bind the GroEL chaperonin platform. The initially soluble FXN-p.Ile154Phe variant partitions onto GroEL more rapidly and is more kinetically liable. These differences in kinetic stability were confirmed using differential scanning fluorimetry. The kinetic and aggregation stability differences of these variants may lead to the distinct functional impairments described in Friedreich's ataxia, the neurodegenerative disease associated to frataxin functional deficiency. This chaperonin platform approach may be useful for identifying small molecule stabilizers since stabilizing ligands to frataxin variants should lead to a concomitant decrease in chaperonin binding.
Subject(s)
Chaperonin 60/metabolism , Iron-Binding Proteins/genetics , Iron-Binding Proteins/metabolism , Calorimetry, Differential Scanning/methods , Escherichia coli/genetics , Humans , Iron-Binding Proteins/chemistry , Kinetics , Methylamines/chemistry , Molecular Probe Techniques , Mutation , Protein Folding , Protein Stability , Solubility , FrataxinABSTRACT
UDCA (ursodeoxycholic acid) is used increasingly for the treatment of cholestatic liver diseases. Among other cytoprotective effects, this endogenous bile acid is a potent inhibitor of apoptosis, interfering with both intrinsic and extrinsic apoptotic pathways. In previous studies, we have demonstrated that the transforming growth factor beta1-induced E2F-1/Mdm2 (murine double minute 2)/p53 apoptotic pathway was an upstream molecular target of UDCA. In agreement with this, we have recently established p53 as a key molecular target in UDCA prevention of cell death. The tumour suppressor p53 is a well-described transcription factor that induces the expression of multiple different pro-apoptotic gene products. Its regulation involves a variety of signalling proteins and small molecules, and occurs at multiple levels, including transcription, translation and post-translation levels. In the present study, by using different biophysical techniques, we have investigated the possibility of a direct interaction between the p53 core domain, also referred to as the DNA-binding domain, and UDCA. Our in vitro analysis did not provide any evidence for direct binding between the bile acid UDCA and the p53 core domain.