Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

Abstract

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan AP2 (ApiAP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical ApiAP2 protein, PF14_0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14_0633 AP2 domain bound to DNA reveals a β-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent α-helix supports the β-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened or eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14_0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the α-helices of the AP2 domains pack against the β-sheets of the dimer mates. DNA-induced dimerization of PF14_0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multi-site binding mode, at least two copies of the consensus sequence recognized by PF14_0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.

TheCryptosporidium parvumApiAP2 gene family: insights into the evolution of apicomplexan AP2 regulatory systems

“…Whether the C. parvum proteins utilize multiple DNA-binding regions simultaneously remains to be determined. Interestingly, C. parvum AP2 domain cgd4_3820 recognizes the sequence 5′-GGTGCACC-3′, while its putative P. falciparum ortholog PFF0200c_D2 (38% identity, with no conservation of residues predicted to be important for base-specific contacts (56)) failed to bind DNA as measured by PBMs. However, a construct of both AP2 domains PFF0200c_D1 (which does show binding) and PFF0200c_D2 joined by a short conserved linker region does bind the same motif as cgd4_3820.…”

Toxoplasma Transcription Factor TgAP2XI-5 Regulates the Expression of Genes Involved in Parasite Virulence and Host Invasion

“…Typical of different apicomplexan AP2 family members (6), the conservation of TgAP2XI-5 with its putative apicomplexan orthologs is restricted primarily to its AP2 domain. This is important because proteins with highly conserved AP2 domains tend to bind to the same DNA motifs, even between organisms of different genera (18,19). It was not surprising, therefore, to find preferential binding of TgAP2XI-5 to a GCTAGC motif within the promoters of T. gondii genes.…”

“…Dimer formation is particularly important because it can alter the temporal nature of transcription factor activities (23), offering one explanation for the lack of correlation between the expression of TgAP2XI-5 and the timed transcription of its putative gene targets. Interestingly, the P. falciparum AP2 protein, PF14_0633, has been shown to form a homodimer upon binding to DNA (18), although in this case dimer formation is probably mediated by disulfide bonds rather than phosphorylation. Yeast two-hybrid screening has also highlighted the potential for heterodimer formation between other AP2s of P. falciparum (24).…”

“…Nevertheless, the TgAP2XI-5 AP2 domain itself was highly conserved between T. gondii and either N. caninum (100% identity), P. falciparum (88.5% identity), or P. berghei (86.5% identity) (Fig. 1b), particularly the three ␤-sheets involved in the direct binding of double-stranded DNA (18,19). Interestingly, despite the apparent expansion in the number of AP2-containing proteins in T. gondii, there did not appear to be any paralogs of TgAP2XI-5, with the most closely related AP2 (TGME49_018960) showing only 40% identity over the DNA-binding domain.…”

The Symbiosis-Related ERN Transcription Factors Act in Concert to Coordinate Rhizobial Host Root Infection

“…The negative effect of this mutation on DNA binding can be adequately explained by 3D homology modeling, based on the earlier solution NMR structure determined for the AP2/ERF domain of AtERF1 interacting with the GCC-box DNA motif (Allen et al, 1998;Yamasaki et al, 2012). ERF TFs exhibit an unusual mode of DNA interaction via three b-strands contacting the DNA backbone and specific bases within the major groove, a global structure that is conserved even in the distantly related apicomplexan ERF-like proteins (Lindner et al, 2010). The T 61 I replacement abolishes a key hydrogen-bond interaction with the sugar-P backbone, in addition to negatively affecting the DNA base interaction of R 37 (Fig.…”