Isolation and characterization of the core single-stranded DNA-binding domain of purine-rich element binding protein B (Purβ)

Abstract

Purβ is a single-stranded nucleic acid-binding protein implicated in the injury-induced repression of genes encoding certain muscle-restricted isoforms of actin and myosin expressed in the heart, skeletal muscle, and vasculature. To better understand how the modular arrangement of the primary sequence of Purβ affects the higher order structure and function of the protein, purified recombinant Purβ was subjected to partial proteolysis in an attempt to identify a well-folded truncation protein that retained purine-rich single-stranded DNA-binding activity. Limited tryptic digestion of Purβ liberated a core ~30 kDa fragment corresponding to residues 29–305 as determined by epitope mapping and mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to self-associate while circular dichroism analysis confirmed that the Purβ core domain is stably folded in the absence of glycine-rich N- and C-terminal sequences. Comparative DNA-binding assays revealed that the isolated core domain interacts with purine-rich cis-elements from the smooth muscle α-actin gene with similar specificity but increased affinity compared to full-length Purβ. These findings suggest that the highly conserved modular repeats of Purβ fold to form a core functional domain, which mediates the specific and high affinity binding of the protein to single-stranded DNA.

Structural Basis of Multisite Single-Stranded DNA Recognition and ACTA2 Repression by Purine-Rich Element Binding Protein B (Purβ)

“…Despite this technical limitation, the Purβ I-II-III core construct did appear to bind PE32-F with comparable affinity and specificity to full-length Purβ under the assay conditions employed (Figure 7). Moreover, the biochemical properties of Purβ I-II-III (residues 41-303) defined in this study are analogous to those of a His tag-free core tryptic fragment of Purβ (residues 29-305) described in an earlier report (40). Interestingly, while the Purβ I-II and Purβ III subdomains each displayed a lower apparent affinity for PE32-F than Purβ I-II-III, the intermolecular subdomain bound more tightly to ssDNA than the intramolecular subdomain.…”

“…Multiple wavelength scans were recorded at 1 nm intervals from 195 to 280 nm on 5.0 μM protein solutions in a 1 mm cuvette at 25°C. Raw CD data were analyzed as previously described (40). …”

“…Importantly, the x-ray crystal structure of a truncated version of Drosophila melanogaster (Dm) Purα (amino acids 40-185) revealed a monomeric Whirly fold-like DNA-binding domain formed by the intramolecular interaction of the first two PUR repeat sequences (39). On the other hand, we recently identified a core tryptic fragment of Purβ (amino acids 29-305) that contains all three PUR repeats, self-associates in the absence of nucleic acid, and retains the ability to interact with the purine-rich strand of the ACTA2 -derived MCAT element with high-affinity and specificity (40). In this study, the putative biological role of Purβ in suppressing ACTA2 expression and restricting myofibroblast cyto-differentiation was first validated via a stable gene knockdown approach.…”

EGFR Fusions as Novel Therapeutic Targets in Lung Cancer

“…As demonstrated by our structural modeling, EGFR-RAD51 fusion proteins could be activated by virtue of RAD51 oligomerization. Similarly, structural modeling has shown that PURB proteins can self-associate in the absence of nucleic acids (28). Additional experimental work will be required to determine whether activation of EGFR fusion proteins is driven by asymmetric dimerization of the EGFR tyrosine kinase domain, dimerization through the known partner oligomerization interface, or both.…”

Structural and functional analysis of single‐nucleotide polymorphic variants of purine‐rich element‐binding protein B

“…Some studies have evaluated components of inflammatory response pathways, but have been met with limited success. 43 This core region of Purβ contains sequence elements termed Pur repeats I, II, and III that are highly conserved in Purα. 14,15 The transcriptional repressor protein, Purβ, is of special interest because of its unique ability to interact with specific ssDNA sequences in the Acta2 promoter and with other transcription factors that dictate Acta2 gene expression in fibroblasts.…”