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Quantitative mapping of binding specificity landscapes for homologous targets by using a high-throughput method.

ABSTRACT: To facilitate investigations of protein-protein interactions (PPIs), we developed a novel platform for quantitative mapping of protein binding specificity landscapes, which combines the multi-target screening of a mutagenesis library into high- and low-affinity populations with sophisticated next-generation sequencing analysis. Importantly, this method generates accurate models to predict affinity and specificity values for any mutation within a protein complex, and requires only a few experimental binding affinity measurements using purified proteins for calibration. We demonstrated the utility of the approach by mapping quantitative landscapes for interactions between the N-terminal domain of the tissue inhibitor of metalloproteinase 2 (N-TIMP2) and three matrix metalloproteinases (MMPs) having homologous structures but different affinities (MMP-1, MMP-3, and MMP-14). The binding landscapes for N-TIMP2/MMP-1 and N-TIMP2/MMP-3 showed the PPIs to be almost fully optimized, with most single mutations giving a loss of affinity. In contrast, the non-optimized PPI for N-TIMP2/MMP-14 was reflected in a wide range of binding affinities, where single mutations exhibited a far more attenuated effect on the PPI. Our new platform reliably and comprehensively identified not only hot- and cold-spot residues, but also specificity-switch mutations that shape target affinity and specificity. Thus, our approach provides a methodology giving an unprecedentedly rich quantitative analysis of the binding specificity landscape, which will broaden the understanding of the mechanisms and evolutionary origins of specific PPIs and facilitate the rational design of specific inhibitors for structurally similar target proteins.

SUBMITTER: Aharon L

PROVIDER: S-EPMC7376575 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Mutational inactivation of plant genes is an essential tool in gene function studies. Plants with inactivated or deleted genes may also be exploited for crop improvement if such mutations/deletions produce a desirable agronomical and/or quality phenotype. However, the use of mutational gene inactivation/deletion has been impeded in polyploid plant species by genetic redundancy, as polyploids contain multiple copies of the same genes (homologous genes) encoded by each of the ancestral genomes. Similar to many other crop plants, bread wheat (Triticum aestivum L.) is polyploid; specifically allohexaploid possessing three progenitor genomes designated as 'A', 'B', and 'D'. Recently modified TILLING protocols have been developed specifically for mutation detection in wheat. Whilst extremely powerful in detecting single nucleotide changes and small deletions, these methods are not suitable for detecting whole gene deletions. Therefore, high-throughput methods for screening of candidate homologous gene deletions are needed for application to wheat populations generated by the use of certain mutagenic agents (e.g. heavy ion irradiation) that frequently generate whole-gene deletions. RESULTS: To facilitate the screening for specific homologous gene deletions in hexaploid wheat, we have developed a TaqMan qPCR-based method that allows high-throughput detection of deletions in homologous copies of any gene of interest, provided that sufficient polymorphism (as little as a single nucleotide difference) amongst homologues exists for specific probe design. We used this method to identify deletions of individual TaPFT1 homologues, a wheat orthologue of the disease susceptibility and flowering regulatory gene PFT1 in Arabidopsis. This method was applied to wheat nullisomic-tetrasomic lines as well as other chromosomal deletion lines to locate the TaPFT1 gene to the long arm of chromosome 5. By screening of individual DNA samples from 4500 M2 mutant wheat lines generated by heavy ion irradiation, we detected multiple mutants with deletions of each TaPFT1 homologue, and confirmed these deletions using a CAPS method. We have subsequently designed, optimized, and applied this method for the screening of homologous deletions of three additional wheat genes putatively involved in plant disease resistance. CONCLUSIONS: We have developed a method for automated, high-throughput screening to identify deletions of individual homologues of a wheat gene. This method is also potentially applicable to other polyploidy plants.

Project description:In this work, we used our recently developed method Spec-seq to characterize the binding specificity of Glucocorticoid receptor in vitro. This GR Spec-seq experiment has been run twice separately (Dec 2014 and Mar 2015) with different library compositions. The basic workflow is the same as our previous work for lac repressor published in Genetics 198.3 (2014): 1329-1343. Recombinant human GR protein was used to facilitate in vitro DNA-binding and separation experiments. Bound and Unbound DNA fragments were separated in EMSA gels, purified, barcoded for further Illumina sequencing. An initial experiment was performed using the putative consensus sequence: AGAACA GGG TGTTCT; randomized library 1: AGAACN NSN NGTTCT [diversity =512]; randomized library 2: AGAANN GGG NNNTCT [diversity = 1024]; randomized library 3: AGAACA GGG TGNNNN [diversity =256]; randomized library 4: AGAACA GGGC NNNTCT [diversity = 64]. The initial total library diversity was ~1853 with a library composition of 10% positive control sequence + randomized libraries 1-4 + 5% negative control sequence. A 5th library containing 2304 sequences based on DDNACW KKN KGTTCT, where D="not C", N="any base", W="A or T" and K="G or T" was subsequently prepared and analyzed using similar ratios of control sequences. Binding conditions for EMSA were 100ng FAM-labelled dsDNA+ 0/0.5/1/2/4uM GR DBD protein for each lane, 1X NEB buffer 4. GR DBD was prepared as previously described. The EMSA was performed using a 9% 33:1 acrylamide gel and TB buffer, and was run at 200V for 30mins @ 0 degrees C. The 2uM protein lane used for final sequencing. Bound/unbound fractions resulting from EMSA of these libraries and conditions were used to generate PWMs as described. The GR PWM that was generated through this analysis was used to define relative binding energies using the patser program (35), which can be accessed online at (http://stormo.wustl.edu/consensus/cgi-bin/Server/Interface/patser.cgi ). Derived binding affinities are proportional to the inverse of the natural log of the calculated energies.

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Quantitative mapping of binding specificity landscapes for homologous targets by using a high-throughput method.

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