An optimized energy potential can predict SH2 domain-peptide interactions

Peptide recognition modules (PRMs) are used throughout biology to mediate protein-protein interactions, and many PRMs are members of large protein domain families. Members of these families are often quite similar to each other, but each domain recognizes a distinct set of peptides, raising the question of how peptide recognition specificity is achieved using similar protein domains. The analysis of individual protein complex structures often gives answers that are not easily applicable to other members of the same PRM family. Bioinformatics-based approaches, one the other hand, may be difficult to interpret physically. Here we integrate structural information with a large, quantitative data set of SH2-peptide interactions to study the physical origin of domain-peptide specificity. We develop an energy model, inspired by protein folding, based on interactions between the amino acid positions in the domain and peptide. We use this model to successfully predict which SH2 domains and peptides interact and uncover the positions in each that are important for specificity. The energy model is general enough that it can be applied to other members of the SH2 family or to new peptides, and the cross-validation results suggest that these energy calculations will be useful for predicting binding interactions. It can also be adapted to study other PRM families, predict optimal peptides for a given SH2 domain, or study other biological interactions, e.g. protein-DNA interactions

ReadOut: structure-based calculation of direct and indirect readout energies and specificities for protein–DNA recognition

Protein–DNA interactions play a central role in regulatory processes at the genetic level. DNA-binding proteins recognize their targets by direct base–amino acid interactions and indirect conformational energy contribution from DNA deformations and elasticity. Knowledge-based approach based on the statistical analysis of protein–DNA complex structures has been successfully used to calculate interaction energies and specificities of direct and indirect readouts in protein–DNA recognition. Here, we have implemented the method as a webserver, which calculates direct and indirect readout energies and Z-scores, as a measure of specificity, using atomic coordinates of protein–DNA complexes. This server is freely available at . The only input to this webserver is the Protein Data Bank (PDB) style coordinate data of atoms or the PDB code itself. The server returns total energy Z-scores, which estimate the degree of sequence specificity of the protein–DNA complex. This webserver is expected to be useful for estimating interaction energy and DNA conformation energy, and relative contributions to the specificity from direct and indirect readout. It may also be useful for checking the quality of protein–DNA complex structures, and for engineering proteins and target DNAs

Predicting Transcription Factor Specificity with All-Atom Models

The binding of a transcription factor (TF) to a DNA operator site can initiate or repress the expression of a gene. Computational prediction of sites recognized by a TF has traditionally relied upon knowledge of several cognate sites, rather than an ab initio approach. Here, we examine the possibility of using structure-based energy calculations that require no knowledge of bound sites but rather start with the structure of a protein-DNA complex. We study the PurR E. coli TF, and explore to which extent atomistic models of protein-DNA complexes can be used to distinguish between cognate and non-cognate DNA sites. Particular emphasis is placed on systematic evaluation of this approach by comparing its performance with bioinformatic methods, by testing it against random decoys and sites of homologous TFs. We also examine a set of experimental mutations in both DNA and the protein. Using our explicit estimates of energy, we show that the specificity for PurR is dominated by direct protein-DNA interactions, and weakly influenced by bending of DNA.Comment: 26 pages, 3 figure

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions

The Hox protein conundrum: The “specifics” of DNA binding for Hox proteins and their partners

Homeotic genes (Hox genes) are homeodomain-transcription factors involved in conferring segmental identity along the anterior-posterior body axis. Molecular characterization of HOX protein function raises some interesting questions regarding the source of the binding specificity of the HOX proteins. How do HOX proteins regulate common and unique target specificity across space and time? This review attempts to summarize and interpret findings in this area, largely focused on results from in vitro and in vivo studies in Drosophila and mouse systems. Recent studies related to HOX protein binding specificity compel us to reconsider some of our current models for transcription factor-DNA interactions. It is crucial to study transcription factor binding by incorporating components of more complex, multi-protein interactions in concert with small changes in binding motifs that can significantly impact DNA binding specificity and subsequent alterations in gene expression. To incorporate the multiple elements that can determine HOX protein binding specificity, we propose a more integrative Cooperative Binding model

Dual-spectral interferometric sensor for quantitative study of protein-DNA interactions

Thesis (Ph.D.)--Boston UniversityThe maintenance and functions of the genome are facilitated by DNA-binding proteins, whose specific binding mechanisms are not yet fully understood. Recently, it was discovered that the recognition and capture ofDNA conformational flexibility and deformation by DNA-binding proteins serve as an indirect readout mechanism for specific recognition and facilitate important cellular functions. Various biophysical techniques have been employed to elucidate this conformational specificity of protein-DNA interactions. These techniques are not sufficiently high-throughput to perform systematic investigation ofvarious protein-DNA complexes and their functions. Microarray-based high-throughput methods enable large-scale and comprehensive evaluation of the binding affmities of protein-DNA interactions, but do not provide conformational information. In this dissertation, we developed a tool that enables high-throughput quantification of both conformational specificity and binding affinity of protein-DNA interactions. Our approach is to combine quantitative detection of DNA conformational change and protein-DNA binding in a DNA microarray format. The DNA conformational change is measured by spectral self-interference fluorescence microscopy that determines surface-immobilized DNA conformation by measuring axial height offluorophores tagged to specific nucleotides. The amount of bound protein and DNA are measured by white light reflectance spectroscopy that quantifies molecular surface densities by measuring bioniolecule layer thicknesses. By implementing a dual-spectral imaging configuration, we can perform the two independent interferometric measurements in parallel using two separate spectral bandwidths. [TRUNCATED

Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family

Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins

Dual-spectral interferometric sensor for quantitative study of protein-DNA interactions

Thesis (Ph.D.)--Boston UniversityThe maintenance and functions of the genome are facilitated by DNA-binding proteins, whose specific binding mechanisms are not yet fully understood. Recently, it was discovered that the recognition and capture ofDNA conformational flexibility and deformation by DNA-binding proteins serve as an indirect readout mechanism for specific recognition and facilitate important cellular functions. Various biophysical techniques have been employed to elucidate this conformational specificity of protein-DNA interactions. These techniques are not sufficiently high-throughput to perform systematic investigation ofvarious protein-DNA complexes and their functions. Microarray-based high-throughput methods enable large-scale and comprehensive evaluation of the binding affmities of protein-DNA interactions, but do not provide conformational information. In this dissertation, we developed a tool that enables high-throughput quantification of both conformational specificity and binding affinity of protein-DNA interactions. Our approach is to combine quantitative detection of DNA conformational change and protein-DNA binding in a DNA microarray format. The DNA conformational change is measured by spectral self-interference fluorescence microscopy that determines surface-immobilized DNA conformation by measuring axial height offluorophores tagged to specific nucleotides. The amount of bound protein and DNA are measured by white light reflectance spectroscopy that quantifies molecular surface densities by measuring bioniolecule layer thicknesses. By implementing a dual-spectral imaging configuration, we can perform the two independent interferometric measurements in parallel using two separate spectral bandwidths. [TRUNCATED

MADS specificity : Unravelling the dual function of the MADS domain protein FRUITFULL

Encrypted in the DNA lays most information needed for the development of an organism. The transcription of this information into precise patterns of gene activity results in the development of different cell types, organs, and developmental structures. Moreover, transcriptional regulation enables an organism to respond to changing environmental conditions. Essential for the regulation of transcription are DNA-binding transcription factors (TFs). TFs bind the DNA in a sequence-specific fashion. Upon binding of a TF to its DNA binding site, TFs typically activate or repress the transcription of nearby genes. To better understand transcriptional regulation it is essential to study DNA binding specificity of TFs. In the last decades, technological advances allowed the development of high-throughput methods to study protein-DNA interactions. Traditional in vitro methods study one or a few interactions, while new high-throughput methods can determine TF specificity by measuring relative DNA-binding affinities against a large collection or even all possible binding sites. Several high-throughput techniques to study TF-DNA interactions are discussed in Chapter 1 of this thesis. These new technologies and methods resulted in a fast growing number of studies on DNA binding specificities of TFs, expanding the knowledge about TF specificity. A review on the current knowledge of TF DNA binding specificity is described in Chapter 1. One aspect that influences DNA binding of TFs are differences in ability to form protein-protein interactions. The aim of this thesis was to study the role of protein-protein interactions in determining DNA binding specificity of a developmental regulatory MADS domain TF in Arabidopsis thaliana. While the members of the MADS-box protein family have many, diverse in vivo functions, all members bind in vitro to a 10-bp motif called the CArG-box. Moreover, studies demonstrated that closely related MADS proteins are expressed in the same cells, therefore encountering the same DNA accessibility and DNA methylation patterns, but bind different in vivo targets. Interestingly, MADS domain proteins bind DNA obligatorily as homo- and heterodimers and the interactions between MADS domain proteins are highly protein specific. Hence, MADS domain proteins are a perfect model system to study the influence of intra-family protein interactions on DNA binding specificity. To study the influence of protein-protein interactions on DNA-binding specificity this work focusses on one specific MADS domain protein, FRUITFULL (FUL). FUL is expressed at two stages during flower development and, in both stages FUL has highly diverse functions. In Chapter 2 we demonstrate using RNA-seq that FUL regulates different sets of target genes in the two stages. Moreover, using ChIP-seq we show that FUL genomic DNA binding is partly tissue-specific. These tissue-specifically bound and regulated genes are in line with the known dual functions of FUL during development. Interestingly, using protein complex immunoprecipitation for the two studied tissues/stages we show that the interactions of FUL with other MADS domain proteins are also tissue-specific. To determine whether the tissue-specific in vivo binding pattern are due to differences in DNA binding specificity of the FUL-MADS dimers, we studied the DNA binding specificities of the different protein complexes using SELEX-seq. The SELEX-seq results show that although all tested dimers preferably bind the canonical binding motif of MADS domain proteins, different dimers have different preferences for nucleotides within and surrounding the canonical binding site. Hence, different MADS domain dimers have different in vitro DNA binding specificities. By mapping the SELEX-seq affinities to the genome we were able to compare these results with in vivo tissue-specific ChIP-seq data. This analysis revealed a strong correlation between tissue-specific dimer affinities and tissue-specific genomic binding sites of FUL. Hence, we show that the choice of MADS dimerization partner influences DNA binding specificity, highlighting the role of intra-family protein interactions in defining DNA binding specificity. To allow other researchers to determine genome-wide DNA binding of TFs Chapter 3 provides a step-by-step guide for ChIP-seq experiments and computational analysis. The protocol is designed for wet-lab biologists to perform ChIP-seq experiments and analyse their own ChIP-seq data. Using the genome-wide DNA binding patterns determined by ChIP-seq, Chapter 4 and Chapter 5 take a more detailed look at some of the genes directly bound by FUL. In Chapter 4, we demonstrate a connection between developmentally and environmentally regulated growth programs. We studied a gene directly bound by FUL in pistil tissue, SMALL AUXIN UPREGULATED RNA 10 (SAUR10). SAUR10 expression is regulated by FUL in multiple tissues, among others cauline leaves, stems, and branches. The results show that the expression of SAUR10 at the abaxial side of branches is influenced by a combination of environmental and developmental regulated growth programs: hormones, light conditions, and FUL binding. This spatial regulation possibly affects the angle between the side branches and the main inflorescence stem. Additionally, we discuss several other FUL target genes involved in hormone pathways and light conditions. Chapter 5 focusses on the putative direct targets of FUL in IM tissue. Among the putative direct targets two genes involved in flavonoid synthesis were identified, FLAVONOID SYNTHESE 1 (FLS1) and UDP-GLUCOSYL TRANSFERASE 78D3 (UGT78D3). Interestingly, similar to the ful-7 mutant, the fls1 mutant is late flowering. Moreover, expression data exposed an increased gene expression for both FLS1 and UGT78D3 in developing meristems and showed FLS1 expression to be influenced by light conditions. We report the first link between the MADS domain protein FUL and flavonoid synthesis in Arabidopsis. Moreover, our results indicate a possible link between flavonoids and flowering time. In Chapter 6 I discuss the findings of this thesis and make suggestions for further research. Taken together, the work in this thesis shows that intra-family protein interactions can influence DNA-binding specificity of a protein. Thereby these protein-protein interactions can influence genome-wide binding patterns and, as a result, the function of a protein. Moreover, by studying several putative direct targets of FUL in more detail, we demonstrated a connection between development and environment in growth-regulated programs. Interestingly, the FUL target SAUR10 is repressed by FUL in several tissues, including cauline leaves, inflorescence stems, and branches. However, no influence of FUL on SAUR10 expression could be detected in the pistil. So, despite the binding of FUL to the promotor of SAUR10 in the pistil, this binding does not result in gene regulation. This finding reflects the complex relation between TF occupancy and gene regulation, further research is needed to better understand this relation. Moreover, besides MADS domain protein interactions, we found FUL to interact with several proteins of other families. The role of these cross-family protein interactions in cooperative gene regulation is not fully understood and will be an important research topic in the coming years.</p