RESUMEN
Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.
RESUMEN
The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring ßα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable ßα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.
Asunto(s)
Guanidina/química , Pliegue de Proteína , Urea/química , Interacciones Hidrofóbicas e Hidrofílicas , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
CCCH-type tandem zinc finger (TZF) domains are found in many RNA-binding proteins (RBPs) that regulate the essential processes of post-transcriptional gene expression and splicing through direct protein-RNA interactions. In Caenorhabditis elegans, RBPs control the translation, stability, or localization of maternal messenger RNAs required for patterning decisions before zygotic gene activation. MEX-5 (Muscle EXcess) is a C. elegans protein that leads a cascade of RBP localization events that is essential for axis polarization and germline differentiation after fertilization. Here, we report that at room temperature, the CCCH-type TZF domain of MEX-5 contains an unstructured zinc finger that folds upon binding of its RNA target. We have characterized the structure and dynamics of the TZF domain of MEX-5 and designed a variant MEX-5 in which both fingers are fully folded in the absence of RNA. Within the thermal range experienced by C. elegans, the population of the unfolded state of the TZF domain of MEX-5 varies. We observe that the TZF domain becomes less disordered at lower temperatures and more disordered at higher temperatures. However, in the temperature range in which C. elegans is fertile, when MEX-5 needs to be functional, only one of the two zinc fingers is folded.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Unión Proteica , ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Dedos de ZincRESUMEN
The human protein TDP-43 is a major component of the cellular aggregates found in amyotrophic lateral sclerosis and other neurodegenerative diseases. Insoluble cytoplasmic aggregates isolated from the brain of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients contain ubiquitinated, hyperphosphorylated, and N-terminally truncated TDP-43. Truncated fragments of TDP-43 identified from patient tissues contain part of the second RNA recognition motif (RRM2) and the disordered C-terminus, indicating that both domains can be involved in aggregation and toxicity. Here, we focus on RRM2. Using all-atom replica-averaged metadynamics simulations with NMR chemical shift restraints, we characterized the atomic structure of non-native states of RRM2, sparsely populated under native conditions. These structures reveal the exposure to the solvent of aggregation-prone peptide regions, normally buried in the native state, supporting a role in aggregation for the partially folded states of RRM2.
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Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Pliegue de Proteína , Motivo de Reconocimiento de ARN , Humanos , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Microdeletion syndromes are genetic diseases caused by multilocus chromosomal deletions too small to be detected by karyotyping. They are typified by complex pleiotropic developmental phenotypes that depend both on the extent of the deletion and variations in genetic background. Microdeletion alleles cause a wide array of consequences involving multiple pathways. How simultaneous haploinsufficiency of numerous adjacent genes leads to complex and variable pleiotropic phenotypes is not well understood. CRISPR/Cas9 genome editing has been shown to induce microdeletion-like alleles at a meaningful rate. Here, we describe a microdeletion allele in Caenorhabditis elegans recovered during a CRISPR/Cas9 genome editing experiment. We mapped the allele to chromosome V, balanced it with a reciprocal translocation crossover suppressor, and precisely defined the breakpoint junction. The allele simultaneously removes 32 protein-coding genes, yet animals homozygous for this mutation are viable as adults. Homozygous animals display a complex phenotype including maternal effect lethality, producing polynucleated embryos that grow into uterine tumors, vulva morphogenesis defects, body wall distensions, uncoordinated movement, and a shortened life span typified by death by bursting. Our work provides an opportunity to explore the complexity and penetrance of microdeletion phenotypes in a simple genetic model system.
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Caenorhabditis elegans , Edición Génica , Animales , Femenino , Fenotipo , Mutación , Caenorhabditis elegans/genética , Deleción CromosómicaRESUMEN
Over the past decade, therapeutic monoclonal antibodies (mAbs) have established their role as valuable agents in the treatment of various diseases ranging from cancers to infectious, cardiovascular and autoimmune diseases. Reactive groups of the amino acids within these proteins make them susceptible to many kinds of chemical modifications during manufacturing, storage and in vivo circulation. Among these reactions, the oxidation of methionine residues to their sulfoxide form is a commonly observed chemical modification in mAbs. When the oxidized methionine is in the complementarity-determining region (CDR), this modification can affect antigen binding and thus abrogate biological activity. For these reasons, it is essential to identify oxidation liabilities during the antibody discovery and development phases. Here, we present an in silico method, based on protein modeling and molecular dynamics simulations, to predict the oxidation-liable residues in the variable region of therapeutic antibodies. Previous studies have used the 2-shell water coordination number descriptor (WCN) to identify methionine residues susceptible to oxidation. Although the WCN descriptor successfully predicted oxidation liabilities when the residue was solvent exposed, the method was much less accurate for partially buried methionine residues. Consequently, we introduce a new descriptor, WCN-OH, that improves the accuracy of prediction of methionine oxidation susceptibility by extending the theoretical framework of the water coordination number to incorporate the effects of polar amino acids side chains in close proximity to the methionine of interest.
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Anticuerpos Monoclonales , Metionina , Metionina/química , Anticuerpos Monoclonales/química , Racemetionina , Oxidación-Reducción , Agua , AminoácidosRESUMEN
Many studies have examined the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on neutralizing antibody activity after they have become dominant strains. Here, we evaluate the consequences of further viral evolution. We demonstrate mechanisms through which the SARS-CoV-2 receptor binding domain (RBD) can tolerate large numbers of simultaneous antibody escape mutations and show that pseudotypes containing up to seven mutations, as opposed to the one to three found in previously studied variants of concern, are more resistant to neutralization by therapeutic antibodies and serum from vaccine recipients. We identify an antibody that binds the RBD core to neutralize pseudotypes for all tested variants but show that the RBD can acquire an N-linked glycan to escape neutralization. Our findings portend continued emergence of escape variants as SARS-CoV-2 adapts to humans.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Evasión Inmune , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Vacuna BNT162/inmunología , Betacoronavirus/inmunología , COVID-19/inmunología , COVID-19/virología , Reacciones Cruzadas , Microscopía por Crioelectrón , Cristalografía por Rayos X , Epítopos , Evolución Molecular , Humanos , Modelos Moleculares , Mutación , Polisacáridos/análisis , Unión Proteica , Dominios Proteicos , Receptores de Coronavirus/química , Receptores de Coronavirus/metabolismo , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Pseudotipado ViralRESUMEN
Tristetraprolin (TTP) and TIS11d are two human RNA-binding proteins that belong to the CCCH-type tandem zinc finger family. In the RNA-free state, TIS11d coordinates a zinc ion in each of its two fingers, while TTP coordinates a single zinc ion with the N-terminal zinc finger. We have previously identified three residues, located in the C-terminal half of a short α-helix in the second zinc finger, that control how structured the RNA-binding domain is in these two proteins: Y151, L152, and Q153 in TTP and H201, T202, and I203 in TIS11d. Here, we have used molecular dynamics, NMR spectroscopy, and other biochemical methods to investigate the role of these three residues in the stability of the RNA-binding domain. We found that the intrahelical hydrogen bond formed by the T202 hydroxyl group in the C-terminal zinc finger of TIS11d is necessary to allow for π-π stacking between the side chains of a conserved phenylalanine and the zinc-coordinating histidine. We demonstrated that the lack of this hydrogen bond in TTP is responsible for the reduced zinc affinity of the C-terminal zinc finger.