Dataset Information

Structure and Dynamics of Stacking Interactions in an Antibody Binding Site.

ABSTRACT: For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of producing Abs with widely varying flexibility. We now report the characterization of the complexes formed between two Abs, 5D11 and 10A6, and the chromophoric ligand that they were evolved to recognize, 8-methoxypyrene-1,3,6-trisulfonic acid (MPTS). The sequences of the Ab genes indicate that they evolved from a common precursor. We also used a variety of spectroscopic methods to probe the photophysics and dynamics of the Ab-MPTS complexes and found that they are similar to each other but distinct from previously characterized anti-MPTS Abs. Structural studies revealed that this difference likely results from a unique mode of binding in which MPTS is sandwiched between the side chain of PheH98, which interacts with the chromophore via T-stacking, and the side chain of TrpL91, which interacts with the chromophore via parallel stacking. The T-stacking interaction appears to mediate relaxation on the picosecond time scale, while the parallel stacking appears to mediate relaxation on an ultrafast, femtosecond time scale, which dominates the response. The anti-MPTS Abs thus not only demonstrate the simultaneous use of the two limiting modes of stacking for molecular recognition, but also provide a unique opportunity to characterize how dynamics might contribute to molecular recognition. Both types of stacking are common in proteins and protein complexes where they may similarly contribute to dynamics and molecular recognition.

SUBMITTER: Adhikary R

PROVIDER: S-EPMC6693866 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Structure and Dynamics of Stacking Interactions in an Antibody Binding Site.

Adhikary Ramkrishna R Zimmermann Jörg J Stanfield Robyn L RL Wilson Ian A IA Yu Wayne W Oda Masayuki M Romesberg Floyd E FE

Biochemistry 20190619 27

For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of produc ...[more]

PMID: 31243995

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Project description:During hematopoiesis, multipotential progenitors differentiate into all the types of cells found in blood by the action of complex gene regulatory networks (GRNs) comprising transcription factor (TF) genes that regulate each other’s expression. The gene regulatory logic—by which we mean the identities of the TF regulators, where they bind, whether they activate or repress, and how they interact with each other—remains unknown for most hematopoietic genes. Here, we utilize high coverage ATAC-Seq and reporters integrated in a site-specific manner to investigate binding-site resolution chromatin accessibility dynamics of Cebpa enhancers during macrophage-neutrophil differentiation. Reporter genes were integrated into the ROSA26 locus using CRISPR/Cas9 in PUER cells, which can be differentiated into neutrophils or macrophages in vitro. Time series reporter data showed an upregulation of two enhancers during the first 48 hours of neutrophil differentiation, which mirrored the temporal expression pattern of the endogenous Cebpa gene. Chromatin accessibility was also profiled at several time points by high coverage (300 million reads per sample) ATAC-Seq. The accessibility patterns were highly correlated between time points, allowing us to pool the data and achieve approximately 50x coverage after filtering. These data revealed that the enhancers had many protected regions of low accessibility interspersed with short high-accessibility islands. All previously characterized TF binding sites and centers of ChIP-Seq peaks overlapped protected regions, indicating that they are TF footprints. Surprisingly, while the total accessibility of the enhancers increased during differentiation, this increase occurred after gene expression had peaked, implying that the total accessibility of the enhancers was not driving gene expression. However, Tn5 counts at the high-accessibility islands adjacent to TF footprints, especially for Cebp sites, increased at earlier time points, suggesting that TF occupancy may drive the upregulation in gene expression as well as the later increase in the total accessibility of the enhancers. Our high coverage accessibility maps reveal a complex regulatory architecture, with 5-7 TFs binding up to 20 sites in a single Cebpa enhancer. Furthermore, the temporal dynamics of accessibility and gene expression do not support a causal role for total accessibility in gene expression. More generally, these results show that profiling gene expression and accessibility at high temporal- and genomic-resolution have the potential to reveal the causal drivers of gene expression changes during development.

Project description:BACKGROUND:The enrichment and importance of some aromatic residues, such as Tyr and Trp, have been widely noticed at the binding interfaces of antibodies from many experimental and statistical results, some of which were even identified as "hot spots" contributing significantly greater to the binding affinity than other amino acids. However, how these aromatic residues influence the immune binding still deserves further investigation. A large-scale examination was done regarding the local spatial environment around the interfacial Tyr or Trp residues. Energetic contribution of these Tyr and Trp residues to the binding affinity was then studied regarding 82 representative antibody interfaces covering 509 immune complexes from the PDB database and IMGT/3Dstructure-DB. RESULTS:The connectivity analysis of interfacial residues showed that Tyr and Trp tended to cluster into the spatial Aromatic Islands (AI) rather than being distributed randomly at the antibody interfaces. Out of 82 antibody-antigen complexes, 72% (59) interfaces were found to contain AI with more than 3 aromatic residues. The statistical test against an empirical distribution indicated that the existence of AI was significant in about 60% representative antibody interfaces. Secondly, the loss of solvent accessible surface area (SASA) for side chains of aromatic residues between actually crowded state and independent state was nicely correlated with the AI size increasing in a linearly positive way which indicated that the aromatic side chains in AI tended to take a compact and ordered stacking conformation at the interfaces. Interestingly, the SASA loss of AI was also correlated roughly with the averaged gap of binding free energy between the theoretical and experimental data for immune complexes. CONCLUSIONS:The results of our study revealed the wide existence and statistical significance of "Aromatic Island" (AI) composed of the spatially clustered Tyr and Trp residues at the antibody interfaces. The regular arrangement and stacking of aromatic side chains in AI could probably produce extra cooperative effects to the binding affinity which was firstly observed through the large-scale data analysis. The finding in this work not only provides insights into the functional role of aromatic residues in the antibody-antigen interaction, but also may facilitate the antibody engineering and potential clinical applications.

Dataset Information

Structure and Dynamics of Stacking Interactions in an Antibody Binding Site.

Publications

Structure and Dynamics of Stacking Interactions in an Antibody Binding Site.

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