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1.
Nat Commun ; 14(1): 1770, 2023 03 30.
Article in English | MEDLINE | ID: mdl-36997531

ABSTRACT

Directed evolution in bacterial or yeast display systems has been successfully used to improve stability and expression of G protein-coupled receptors for structural and biophysical studies. Yet, several receptors cannot be tackled in microbial systems due to their complex molecular composition or unfavorable ligand properties. Here, we report an approach to evolve G protein-coupled receptors in mammalian cells. To achieve clonality and uniform expression, we develop a viral transduction system based on Vaccinia virus. By rational design of synthetic DNA libraries, we first evolve neurotensin receptor 1 for high stability and expression. Second, we demonstrate that receptors with complex molecular architectures and large ligands, such as the parathyroid hormone 1 receptor, can be readily evolved. Importantly, functional receptor properties can now be evolved in the presence of the mammalian signaling environment, resulting in receptor variants exhibiting increased allosteric coupling between the ligand binding site and the G protein interface. Our approach thus provides insights into the intricate molecular interplay required for GPCR activation.


Subject(s)
Vaccinia , Animals , Ligands , Vaccinia virus/genetics , Vaccinia virus/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/genetics , Mammals/metabolism
3.
PLoS Genet ; 12(11): e1006321, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27898685

ABSTRACT

The active sites of multisubunit RNA polymerases have a "trigger loop" (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.


Subject(s)
Mutant Proteins/genetics , RNA Polymerase II/genetics , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Alleles , Catalysis , Catalytic Domain/genetics , Crystallography, X-Ray , Mutant Proteins/chemistry , Mutation , Protein Folding , Protein Structure, Secondary , Protein Transport/genetics , RNA Polymerase II/chemistry , Saccharomyces cerevisiae/enzymology , Substrate Specificity
4.
MAbs ; 8(1): 176-86, 2016.
Article in English | MEDLINE | ID: mdl-26406987

ABSTRACT

X-ray structure analysis of 4 antibody Fab fragments, each in complex with human granulocyte macrophage colony stimulating factor (GM-CSF), was performed to investigate the changes at the protein-protein binding interface during the course of in vitro affinity maturation by phage display selection. The parental antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized), MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All antibodies bind to a conformational epitope that can be divided into 3 sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF N-terminus (residues 11-24), a short second sub-epitope (residues 83-89) and a third at the C-terminus (residues 112-123). Modifications introduced during affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional hydrogen bonds and van der Waals contacts, respectively, providing a rationale for the observed improvement in binding affinity and neutralization potency. Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash with GM-CSF binding to GM-CSF receptor α and ß chain. This predicted mutually exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding inhibition assay. Finally, high throughput sequencing of clones obtained after affinity maturation phage display pannings revealed highly selected consensus sequences for CDR-H2 as well for CDR-L3, which are in accordance with the sequence of the highest affinity antibody MOR04357. The resolved crystal structures highlight the criticality of these strongly selected residues for high affinity interaction with GM-CSF.


Subject(s)
Antibodies, Monoclonal , Antibodies, Neutralizing , Antibody Affinity , Directed Molecular Evolution , Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Humans
5.
PLoS Genet ; 10(3): e1004157, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24651573

ABSTRACT

Active and repressed ribosomal RNA (rRNA) genes are characterised by specific epigenetic marks and differentially positioned nucleosomes at their promoters. Repression of the rRNA genes requires a non-coding RNA (pRNA) and the presence of the nucleolar remodeling complex (NoRC). ATP-dependent chromatin remodeling enzymes are essential regulators of DNA-dependent processes, and this regulation occurs via the modulation of DNA accessibility in chromatin. We have studied the targeting of NoRC to the rRNA gene promoter; its mechanism of nucleosome positioning, in which a nucleosome is placed over the transcription initiation site; and the functional role of the pRNA. We demonstrate that NoRC is capable of recognising and binding to the nucleosomal rRNA gene promoter on its own and binds with higher affinity the nucleosomes positioned at non-repressive positions. NoRC recognises the promoter nucleosome within a chromatin array and positions the nucleosomes, as observed in vivo. NoRC uses the release mechanism of positioning, which is characterised by a reduced affinity for the remodeled substrate. The pRNA specifically binds to NoRC and regulates the enzyme by switching off its ATPase activity. Given the known role of pRNA in tethering NoRC to the rDNA, we propose that pRNA is a key factor that links the chromatin modification activity and scaffolding function of NoRC.


Subject(s)
Chromatin Assembly and Disassembly/genetics , Epigenesis, Genetic , RNA, Ribosomal/genetics , RNA, Untranslated/genetics , Acetylation , Adenosine Triphosphatases/metabolism , Animals , Chromatin/genetics , Histones/genetics , Mice , Multiprotein Complexes/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nucleosomes/genetics , Nucleosomes/metabolism , Promoter Regions, Genetic
6.
MAbs ; 5(3): 445-70, 2013.
Article in English | MEDLINE | ID: mdl-23571156

ABSTRACT

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Immunoglobulin G/metabolism , Immunotherapy , Antibodies, Monoclonal/genetics , Antibody Affinity , Cells, Cultured , Complementarity Determining Regions/genetics , Dimerization , Drug Design , Gene Expression , Gene Library , Humans , Immunoglobulin G/genetics , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Protein Engineering , Protein Stability
7.
J Mol Biol ; 422(3): 414-28, 2012 Sep 21.
Article in English | MEDLINE | ID: mdl-22683350

ABSTRACT

To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution.


Subject(s)
Detergents/chemistry , Detergents/metabolism , Micelles , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Animals , Cell Membrane/genetics , Cell Membrane/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Gene Library , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Models, Molecular , Mutation , Protein Conformation , Rats , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/metabolism , Receptors, Neurotensin/genetics , Receptors, Neurotensin/metabolism , Recombination, Genetic
8.
J Mol Biol ; 412(1): 55-71, 2011 Sep 09.
Article in English | MEDLINE | ID: mdl-21787786

ABSTRACT

We present a method for synthetic antibody library generation that combines the use of high-throughput immune repertoire analysis and a novel synthetic technology. The library design recapitulates positional amino acid frequencies observed in natural antibody repertoires. V-segment diversity in four heavy (V(H)) and two kappa (V(κ)) germlines was introduced based on the analysis of somatically hypermutated donor-derived repertoires. Complementarity-determining region 3 length and amino acid designs were based on aggregate frequencies of all V(H) and V(κ) sequences in the data set. The designed libraries were constructed through an adaptation of a novel gene synthesis technology that enables precise positional control of amino acid composition and incorporation frequencies. High-throughput pyrosequencing was used to monitor the fidelity of construction and characterize genetic diversity in the final 3.6×10(10) transformants. The library exhibited Fab expression superior to currently reported synthetic approaches of equivalent diversity, with greater than 93% of clones observed to successfully display both a correctly folded heavy chain and a correctly folded light chain. Genetic diversity in the library was high, with 95% of 7.0×10(5) clones sequenced observed only once. The obtained library diversity explores a comparable sequence space as the donor-derived natural repertoire and, at the same time, is able to access novel recombined diversity due to lack of segmental linkage. The successful isolation of low- and subnanomolar-affinity antibodies against a diverse panel of receptors, growth factors, enzymes, antigens from infectious reagents, and peptides confirms the functional viability of the design strategy.


Subject(s)
Antibodies/chemistry , Peptide Library , Biosensing Techniques , Enzyme-Linked Immunosorbent Assay , Genetic Variation , Humans , Models, Theoretical
9.
Proc Natl Acad Sci U S A ; 104(40): 15635-40, 2007 Oct 02.
Article in English | MEDLINE | ID: mdl-17893337

ABSTRACT

Chromatin-remodeling complexes can translocate nucleosomes along the DNA in an ATP-coupled reaction. This process is an important regulator of all DNA-dependent processes because it determines whether certain DNA sequences are found in regions between nucleosomes with increased accessibility for other factors or wrapped around the histone octamer complex. In a comparison of seven different chromatin-remodeling machines (ACF, ISWI, Snf2H, Chd1, Mi-2, Brg1, and NURF), it is demonstrated that these complexes can read out DNA sequence features to establish specific nucleosome-positioning patterns. For one of the remodelers, ACF, we identified a 40-bp DNA sequence element that directs nucleosome positioning. Furthermore, we show that nucleosome positioning by the remodelers ACF and Chd1 is determined by a reduced affinity to the end product of the translocation reaction. The results suggest that the linkage of differential remodeling activities with the intrinsic binding preferences of nucleosomes can result in establishing distinct chromatin structures that depend on the DNA sequence and define the DNA accessibility for other protein factors.


Subject(s)
Chromatin/physiology , DNA/chemistry , Nucleic Acid Conformation , Nucleosomes/physiology , Animals , Base Sequence , Chromatin/ultrastructure , DNA/genetics , Mammals , Nucleosomes/ultrastructure
10.
Nat Struct Mol Biol ; 12(8): 683-90, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16025127

ABSTRACT

The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. We analyzed the ACF-nucleosome interactions and determined the characteristics of ACF-dependent nucleosome remodeling. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy measurements show that ACF can bind four DNA duplexes simultaneously in a complex that contains two Acf1 and ISWI molecules. Using bead-bound nucleosomal substrates, nucleosome movement by mechanisms involving DNA twisting was excluded. Furthermore, an ACF-dependent local detachment of DNA from the nucleosome was demonstrated in a novel assay based on the preferred intercalation of ethidium bromide to free DNA. The findings suggest a loop recapture mechanism in which ACF introduces a DNA loop at the nucleosomal entry site that propagates over the histone octamer surface and leads to nucleosome repositioning.


Subject(s)
Chromatin Assembly and Disassembly/physiology , DNA/metabolism , Drosophila Proteins/metabolism , Models, Genetic , Nucleosomes/metabolism , Transcription Factors/metabolism , Adenosine Triphosphatases/metabolism , Animals , DNA Footprinting , Drosophila , Electrophoretic Mobility Shift Assay , Ethidium , Histones/metabolism , Nucleosomes/physiology , Polymerase Chain Reaction , Spectrometry, Fluorescence
11.
Nucleic Acids Res ; 32(14): 4091-9, 2004.
Article in English | MEDLINE | ID: mdl-15292447

ABSTRACT

The transcription termination factor (TTF)-I is a multifunctional nucleolar protein that terminates ribosomal gene transcription, mediates replication fork arrest and regulates RNA polymerase I transcription on chromatin. TTF-I plays a dual role in rDNA regulation, being involved in both activation and silencing of rDNA transcription. The N-terminal part of TTF-I contains a negative regulatory domain (NRD) that inhibits DNA binding. Here we show that interactions between the NRD and the C-terminal part of TTF-I mask the DNA-binding domain of TTF-I. However, interaction with TIP5, a subunit of the nucleolar chromatin remodeling complex, NoRC, recovers DNA-binding activity. We have mapped the protein domains that mediate the interaction between TTF-I and TIP5. The association of TIP5 with the NRD facilitates DNA binding of TTF-I and leads to the recruitment of NoRC to the rDNA promoter. Thus, TTF-I and NoRC act in concert to silence rDNA transcription.


Subject(s)
Chromatin Assembly and Disassembly , DNA-Binding Proteins/metabolism , Gene Silencing , Genes, rRNA , Animals , Chromosomal Proteins, Non-Histone/metabolism , DNA/metabolism , DNA-Binding Proteins/chemistry , HeLa Cells , Humans , Macromolecular Substances , Mice , NIH 3T3 Cells , Protein Structure, Tertiary , Protein Subunits/metabolism , RNA Polymerase I/antagonists & inhibitors , Transcription Factors , Transcription, Genetic
12.
Mol Cell Biol ; 24(4): 1791-8, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14749393

ABSTRACT

The rRNA gene cluster consists of multiple transcription units. Half of these are active, while the other half are transcriptionally inactive. Previously, in vivo studies have demonstrated that silencing of ribosomal DNA (rDNA) is mediated by the chromatin remodeling NoRC (nucleolar remodeling complex). To explore the mechanisms underlying NoRC-directed silencing of rDNA transcription, we investigated the effect of recombinant NoRC on RNA polymerase I transcription on reconstituted chromatin templates. We show that NoRC interacts with the transcription terminator factor (TTF-I), and this interaction is required both for the binding of TTF-I to its promoter-proximal target site and for the recruitment of NoRC to the promoter. After association with the rDNA promoter, NoRC alters the position of the promoter-bound nucleosome, thereby repressing RNA polymerase I transcription. This NoRC-directed rDNA repression requires the N terminus of histone H4. Repression is effective before preinitiation complex formation and as such is unable to exert an effect upon activated rDNA genes. Furthermore, the early steps of rDNA repression do not depend on DNA and histone modifications. These results reveal an important role for TTF-I in recruiting NoRC to rDNA and an active role for NoRC in the establishment of rDNA silencing.


Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA, Ribosomal/genetics , Gene Silencing , Repressor Proteins/metabolism , Acetylation , Adenosine Triphosphatases/genetics , Animals , Cell Line , Chromatin/metabolism , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/genetics , DNA Methylation , DNA, Ribosomal/chemistry , DNA, Ribosomal/metabolism , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , DNA-Binding Proteins/metabolism , Histones/chemistry , Histones/metabolism , Kinetics , Macromolecular Substances , Promoter Regions, Genetic/genetics , Protein Binding , RNA Polymerase I/metabolism , Repressor Proteins/genetics , Templates, Genetic , Transcription Factors , Transcription, Genetic/genetics
13.
EMBO J ; 21(24): 6865-73, 2002 Dec 16.
Article in English | MEDLINE | ID: mdl-12486007

ABSTRACT

Nucleosome remodelling complexes CHRAC and ACF contribute to chromatin dynamics by converting chemical energy into sliding of histone octamers on DNA. Their shared ATPase subunit ISWI binds DNA at the sites of entry into the nucleosome. A prevalent model assumes that DNA distortions catalysed by ISWI are converted into relocation of DNA relative to a histone octamer. HMGB1, one of the most abundant nuclear non-histone proteins, binds with preference to distorted DNA. We have now found that transient interaction of HMGB1 with nucleosomal linker DNA overlapping ISWI-binding sites enhances the ability of ACF to bind nucleosomal DNA and accelerates the sliding activity of limiting concentrations of remodelling factor. By contrast, an HMGB1 mutant with increased binding affinity was inhibitory. These observations are consistent with a role for HMGB1 as a DNA chaperone facilitating the rate-limiting DNA distortion during nucleosome remodelling.


Subject(s)
Chromatin/metabolism , DNA/metabolism , HMGB1 Protein/metabolism , Mutation , Nucleosomes/metabolism , Recombinant Proteins/metabolism , Animals , Binding Sites , DNA Footprinting , Dose-Response Relationship, Drug , Drosophila , Mice , Models, Biological , Protein Binding , Time Factors
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