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1.
Commun Biol ; 6(1): 997, 2023 09 29.
Article in English | MEDLINE | ID: mdl-37773269

ABSTRACT

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1ß antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1ß complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.


Subject(s)
Antibodies , Interleukin-17 , Epitopes/chemistry , Antigens , Binding Sites, Antibody
2.
Nucleic Acids Res ; 51(11): e61, 2023 06 23.
Article in English | MEDLINE | ID: mdl-37014016

ABSTRACT

Deep parallel sequencing (NGS) is a viable tool for monitoring scFv and Fab library dynamics in many antibody engineering high-throughput screening efforts. Although very useful, the commonly used Illumina NGS platform cannot handle the entire sequence of scFv or Fab in a single read, usually focusing on specific CDRs or resorting to sequencing VH and VL variable domains separately, thus limiting its utility in comprehensive monitoring of selection dynamics. Here we present a simple and robust method for deep sequencing repertoires of full length scFv, Fab and Fv antibody sequences. This process utilizes standard molecular procedures and unique molecular identifiers (UMI) to pair separately sequenced VH and VL. We show that UMI assisted VH-VL matching allows for a comprehensive and highly accurate mapping of full length Fv clonal dynamics in large highly homologous antibody libraries, as well as identification of rare variants. In addition to its utility in synthetic antibody discovery processes, our method can be instrumental in generating large datasets for machine learning (ML) applications, which in the field of antibody engineering has been hampered by conspicuous paucity of large scale full length Fv data.


Subject(s)
Gene Library , Single-Chain Antibodies , Immunoglobulin Heavy Chains/genetics , Single-Chain Antibodies/genetics , High-Throughput Nucleotide Sequencing , Machine Learning
3.
Cell Rep ; 25(8): 2121-2131.e5, 2018 11 20.
Article in English | MEDLINE | ID: mdl-30463010

ABSTRACT

The ultimate goal of protein design is to introduce new biological activity. We propose a computational approach for designing functional antibodies by focusing on functional epitopes, integrating large-scale statistical analysis with multiple structural models. Machine learning is used to analyze these models and predict specific residue-residue contacts. We use this approach to design a functional antibody to counter the proinflammatory effect of the cytokine interleukin-17A (IL-17A). X-ray crystallography confirms that the designed antibody binds the targeted epitope and the interaction is mediated by the designed contacts. Cell-based assays confirm that the antibody is functional. Importantly, this approach does not rely on a high-quality 3D model of the designed complex or even a solved structure of the target. As demonstrated here, this approach can be used to design biologically active antibodies, removing some of the main hurdles in antibody design and in drug discovery.


Subject(s)
Antibodies/immunology , Antibody Specificity/immunology , Computational Biology/methods , Epitopes/chemistry , Algorithms , Amino Acid Sequence , Antibodies/chemistry , Humans , Immunoglobulin Fab Fragments/chemistry , Models, Molecular
4.
Curr Opin Struct Biol ; 51: 156-162, 2018 08.
Article in English | MEDLINE | ID: mdl-29791878

ABSTRACT

Antibody design aims to create new antibodies with biological activity that can be used in therapy and research. Traditional methods for antibody discovery, such as animal immunization and large-scale library screening, generate antibodies that bind to the target of interest, but do not necessarily have the desired functional effect. Computational methods can be utilized as a means to guide the search for biologically relevant antibodies, focusing on specificity and affinity determinants to target a particular region of the antigen. Such an approach would allow for the design of epitope-specific antibodies that will have the desired effect on the function of the targeted protein.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Drug Design , Protein Engineering , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/pharmacology , Antibody Affinity , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/metabolism , Antigens/chemistry , Antigens/immunology , Epitopes/chemistry , Epitopes/immunology , Humans , Models, Molecular , Molecular Conformation , Protein Binding , Structure-Activity Relationship
5.
J Comput Aided Mol Des ; 24(12): 971-91, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20976528

ABSTRACT

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Ion Transport/drug effects , Protein Folding/drug effects , Animals , Binding Sites/genetics , Cell Line , Cells, Cultured , Computer Simulation , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis/physiopathology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , HeLa Cells , High-Throughput Screening Assays , Humans , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Rats , Rats, Inbred F344 , Respiratory Mucosa/drug effects , Sequence Deletion , Small Molecule Libraries/chemistry , Structure-Activity Relationship
6.
J Biol Chem ; 285(46): 35825-35, 2010 Nov 12.
Article in English | MEDLINE | ID: mdl-20667826

ABSTRACT

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Mutation , Phenylalanine/genetics , Binding Sites/genetics , Blotting, Western , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Models, Molecular , Phenylalanine/chemistry , Phenylalanine/metabolism , Protein Binding , Protein Folding , Protein Structure, Tertiary , Protein Transport , Sequence Deletion , Suppression, Genetic
7.
Protein Sci ; 14(11): 2849-61, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16251366

ABSTRACT

The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6-kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four alpha-helices and a mixed beta-sheet consisting of four parallel and anti-parallel beta-strands, where the alpha-helices sandwich the beta-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii, and Sulfolobus solfataricus reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Data Bank but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good-quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal, and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.


Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/classification , Archaeoglobus fulgidus/enzymology , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/classification , Models, Molecular , Archaea/classification , Archaea/enzymology , Archaeoglobus fulgidus/classification , Bacteria/classification , Bacteria/enzymology , Binding Sites , Conserved Sequence , Evolution, Molecular , Humans , Nuclear Magnetic Resonance, Biomolecular , Phylogeny , Sequence Homology, Amino Acid , Solutions , Structural Homology, Protein
9.
J Mol Biol ; 344(2): 549-65, 2004 Nov 19.
Article in English | MEDLINE | ID: mdl-15522304

ABSTRACT

The isc and suf operons in Escherichia coli represent alternative genetic systems optimized to mediate the essential metabolic process of iron-sulfur cluster (Fe-S) assembly under basal or oxidative-stress conditions, respectively. Some of the proteins in these two operons share strong sequence homology, e.g. the cysteine desulfurases IscS and SufS, and presumably play the same role in the oxygen-sensitive assembly process. However, other proteins in these operons share no significant homology and occur in a mutually exclusive manner in Fe-S assembly operons in other organisms (e.g. IscU and SufE). These latter proteins presumably play distinct roles adapted to the different assembly mechanisms used by the two systems. IscU has three invariant cysteine residues that function as a template for Fe-S assembly while accepting a sulfur atom from IscS. SufE, in contrast, does not function as an Fe-S assembly template but has been suggested to function as a shuttle protein that uses a persulfide linkage to a single invariant cysteine residue to transfer a sulfur atom from SufS to an alternative Fe-S assembly template. Here, we present and analyze the 2.0A crystal structure of E.coli SufE. The structure shows that the persulfide-forming cysteine occurs at the tip of a loop with elevated B-factors, where its side-chain is buried from solvent exposure in a hydrophobic cavity located beneath a highly conserved surface. Despite the lack of sequence homology, the core of SufE shows strong structural similarity to IscU, and the sulfur-acceptor site in SufE coincides with the location of the cysteine residues mediating Fe-S cluster assembly in IscU. Thus, a conserved core structure is implicated in mediating the interactions of both SufE and IscU with the mutually homologous cysteine desulfurase enzymes present in their respective operons. A similar core structure is observed in a domain found in a variety of Fe-S cluster containing flavoenzymes including xanthine dehydrogenase, where it also mediates interdomain interactions. Therefore, the core fold of SufE/IscU has been adapted to mediate interdomain interactions in diverse redox protein systems in the course of evolution.


Subject(s)
Conserved Sequence/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Sulfur/metabolism , Amino Acid Sequence , Carbon-Sulfur Lyases/chemistry , Carbon-Sulfur Lyases/genetics , Carbon-Sulfur Lyases/metabolism , Crystallography, X-Ray , Cysteine/chemistry , Escherichia coli/chemistry , Escherichia coli Proteins/genetics , Evolution, Molecular , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Iron-Sulfur Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Operon , Oxidation-Reduction , Phylogeny , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Solutions , Spectrum Analysis, Raman , Xanthine Dehydrogenase/chemistry
10.
J Mol Biol ; 344(2): 567-83, 2004 Nov 19.
Article in English | MEDLINE | ID: mdl-15522305

ABSTRACT

IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Zinc/metabolism , Amino Acid Sequence , Binding Sites , Chelating Agents/pharmacology , Conserved Sequence , Cysteine/chemistry , Cysteine/metabolism , Evolution, Molecular , Haemophilus influenzae/chemistry , Histidine/chemistry , Histidine/metabolism , Hydrogen Bonding , Ligands , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Solutions , Spectrum Analysis, Raman
13.
Protein Sci ; 12(12): 2823-30, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14627742

ABSTRACT

We report NMR assignments and solution structure of the 71-residue 30S ribosomal protein S28E from the archaean Pyrococcus horikoshii, target JR19 of the Northeast Structural Genomics Consortium. The structure, determined rapidly with the aid of automated backbone resonance assignment (AutoAssign) and automated structure determination (AutoStructure) software, is characterized by a four-stranded beta-sheet with a classic Greek-key topology and an oligonucleotide/oligosaccharide beta-barrel (OB) fold. The electrostatic surface of S28E exhibits positive and negative patches on opposite sides, the former constituting a putative binding site for RNA. The 13 C-terminal residues of the protein contain a consensus sequence motif constituting the signature of the S28E protein family. Surprisingly, this C-terminal segment is unstructured in solution.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Pyrococcus horikoshii/chemistry , Ribosomal Proteins/chemistry , Amino Acid Sequence , Models, Molecular , Molecular Sequence Data , Sequence Alignment
14.
Proteins ; 53 Suppl 6: 430-5, 2003.
Article in English | MEDLINE | ID: mdl-14579332

ABSTRACT

We participated in the fold recognition and homology sections of CASP5 using primarily in-house software. The central feature of our structure prediction strategy involved the ability to generate good sequence-to-structure alignments and to quickly transform them into models that could be evaluated both with energy-based methods and manually. The in-house tools we used include: a) HMAP (Hybrid Multidimensional Alignment Profile)-a profile-to-profile alignment method that is derived from sequence-enhanced multiple structure alignments in core regions, and sequence motifs in non-structurally conserved regions. b) NEST-a fast model building program that applies an "artificial evolution" algorithm to construct a model from a given template and alignment. c) GRASP2-a new structure and alignment visualization program incorporating multiple structure superposition and domain database scanning modules. These methods were combined with model evaluation based on all atom and simplified physical-chemical energy functions. All of these methods were under development during CASP5 and consequently a great deal of manual analysis was carried out at each stage of the prediction process. This interactive model building procedure has several advantages and suggests important ways in which our and other methods can be improved, examples of which are provided.


Subject(s)
Protein Folding , Proteins/chemistry , Sequence Alignment/methods , Algorithms , Amino Acid Sequence , Binding Sites/genetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Proteins/genetics , Sequence Homology, Amino Acid , Thermodynamics
15.
Protein Sci ; 12(9): 1813-21, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12930981

ABSTRACT

The success of structural genomics initiatives requires the development and application of tools for structure analysis, prediction, and annotation. In this paper we review recent developments in these areas; specifically structure alignment, the detection of remote homologs and analogs, homology modeling and the use of structures to predict function. We also discuss various rationales for structural genomics initiatives. These include the structure-based clustering of sequence space and genome-wide function assignment. It is also argued that structural genomics can be integrated into more traditional biological research if specific biological questions are included in target selection strategies.


Subject(s)
Genome , Proteins/chemistry , Proteome , Amino Acid Motifs , Animals , Computer Simulation , Databases as Topic , Humans , Models, Theoretical , Phylogeny , Protein Conformation , Structure-Activity Relationship
17.
Protein Sci ; 12(7): 1556-61, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12824501

ABSTRACT

The structure of Vibrio cholerae protein VC0424 was determined by NMR spectroscopy. VC0424 belongs to a conserved family of bacterial proteins of unknown function (COG 3076). The structure has an alpha-beta sandwich architecture consisting of two layers: a four-stranded antiparallel beta-sheet and three side-by-side alpha-helices. The secondary structure elements have the order alphabetaalphabetabetaalphabeta along the sequence. This fold is the same as the ferredoxin-like fold, except with an additional long N-terminal helix, making it a variation on this common motif. A cluster of conserved surface residues on the beta-sheet side of the protein forms a pocket that may be important for the biological function of this conserved family of proteins.


Subject(s)
Ferredoxins/chemistry , Vibrio cholerae/chemistry , Amino Acid Sequence , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Conformation , Sequence Alignment , Solutions
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