Solution structure of the RNA binding domain in the human muscleblind‐like protein 2

The muscleblind‐like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1–4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre‐mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH‐type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel β‐sheet with the N‐terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three‐dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH‐type zinc finger motifs in the MBNL protein family.     

Subsets of the zinc finger motifs in dsRBP-ZFa can bind double-stranded RNA

dsRBP-ZFa is a Xenopus zinc finger protein that binds dsRNA and RNA-DNA hybrids with high affinity and in a sequence-independent manner. The protein consists of a basic N-terminal region with seven C2H2 zinc finger motifs and an acidic C-terminal region that is not required for binding. The last four zinc finger motifs, and the linkers that join them, are nearly identical repeats, while the first three motifs and their linkers are each unique. To identify which regions of the protein are involved in nucleic acid binding, we examined the ability of five protein fragments to bind dsRNA and RNA-DNA hybrids. Our studies reveal that a fragment encompassing the three N-terminal, unique zinc finger motifs and another encompassing the last three of the nearly identical motifs have binding properties similar to the full-length protein. Since these two fragments do not share zinc finger motifs of the same sequence, dsRBP-ZFa must contain more than one type of zinc finger motif capable of binding dsRNA. As with the full-length protein, ssRNA and DNA do not significantly compete for dsRNA binding by the fragments.     

Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affinities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-higher affinity for the WTE sequence compared with that of the EGR-1 binding motif. Mutational analysis of the WTE motif revealed a significant contribution to binding affinity by the adenine nucleotide at the eighth position (5'GCGTGGGAGT3') as well as by the 3'-most thymine (5'GCGTGGGAGT3'), whereas mutations in either flanking nucleotides or other nucleotides in the core sequence did not significantly affect the specific binding affinity. Mutations within WT1 zinc fingers II to IV abolished the sequence-specific binding of WT1 to WTE, whereas alterations within the first WT1 zinc finger reduced the binding affinity approximately 10-fold but did not abolish sequence recognition. We have thus identified a WT1 target, which, although similar in sequence to the EGR-1 motif, shows a 20- to 30-fold-higher affinity for WT1. These results suggest that physiological action of WT1 is mediated by binding sites of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is discussed in the context of recent structural analysis.     

Solution structure of the PHD finger from the human KIAA1045 protein

Cross‐brace structural motifs are required as a scaffold to design artificial RING fingers (ARFs) that function as ubiquitin ligase (E3) in ubiquitination and have specific ubiquitin‐conjugating enzyme (E2)‐binding capabilities. The Simple Modular Architecture Research Tool database predicted the amino acid sequence 131–190 (KIAA1045ZF) of the human KIAA1045 protein as an unidentified structural region. Herein, the stoichiometry of zinc ions estimated spectrophotometrically by the metallochromic indicator revealed that the KIAA1045ZF motif binds to two zinc atoms. The structure of the KIAA1045ZF motif bound to the zinc atoms was elucidated at the atomic level by nuclear magnetic resonance. The actual structure of the KIAA1045ZF motif adopts a C₄HC₃‐type PHD fold belonging to the cross‐brace structural family. Therefore, the utilization of the KIAA1045ZF motif as a scaffold may lead to the creation of a novel ARF.