X-ray crystallographic and NMR studies of protein–protein and protein–nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2

Du, Zhihua; Lee, John K.; Fenn, Sebastian; Tjhen, Richard; Stroud, Robert M.; James, Thomas Leroy

doi:10.1261/rna.410107

Cited by 57 publications

(80 citation statements)

References 33 publications

(34 reference statements)

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“…3B). This assignment is consistent with homology modeling of IMP1 KH3 with hnRNP K KH3, PCBP2 KH1, and Nova-2 KH3, all of which specifically recognize a cytosine by base-specific hydrogen bonds made by an arginine residue located within the third b strand of their KH domains (Lewis et al 2000;Backe et al 2005;Du et al 2007Du et al , 2008. Addition of the 5RE RNA to KH34 resulted in the disappearance of many amide resonances within the canonical RNA-binding surface of KH4 as well as smaller chemical shift differences that were located within KH3 (Fig.…”

Section: Resultssupporting

confidence: 73%

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

Patel¹,

Mitra²,

Harris³

et al. 2012

Genes Dev.

150

196

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How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the b-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 59 element (CGGAC) followed by a variable 39 element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34.

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Section: Resultssupporting

confidence: 73%

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

Patel¹,

Mitra²,

Harris³

et al. 2012

Genes Dev.

150

196

View full text Add to dashboard Cite

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“…The functional implication is that individual domains might either act independently or cooperatively, for example, to increase nucleic acid recognition (Lunde et al 2007). Further clustering might also be achieved by the tendency of individual KH domains to self-associate (Du et al 2007;Valverde et al 2008). However, these homo-oligomerization properties have generally been extrapolated from biochemical and crystallographic studies of protein fragments containing either one or two KH domains.…”

Section: Introductionmentioning

confidence: 99%

Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform

Nakel¹,

Hartung²,

Bonneau³

et al. 2010

RNA

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Caenorhabditis elegans GLD-3 is a five K homology (KH) domain-containing protein involved in the translational control of germline-specific mRNAs during embryogenesis. GLD-3 interacts with the cytoplasmic poly(A)-polymerase GLD-2. The two proteins cooperate to recognize target mRNAs and convert them into a polyadenylated, translationally active state. We report the 2.8-Å -resolution crystal structure of a proteolytically stable fragment encompassing the KH2, KH3, KH4, and KH5 domains of C. elegans GLD-3. The structure reveals that the four tandem KH domains are organized into a globular structural unit. The domains are involved in extensive side-by-side interactions, similar to those observed in previous structures of dimeric KH domains, as well as head-to-toe interactions. Small-angle X-ray scattering reconstructions show that the N-terminal KH domain (KH1) forms a thumb-like protrusion on the KH2-KH5 unit. Although KH domains are putative RNA-binding modules, the KH region of GLD-3 is unable in isolation to cross-link RNA. Instead, the KH1 domain mediates the direct interaction with the poly(A)-polymerase GLD-2, pointing to a function of the KH region as a protein-protein interaction platform.

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“…see Ref. 28), knowledge about how PCBPs engage in protein-protein interaction is also very limited. While a protein-D/RNA interaction seems essential for initiating the sequence of events, protein-protein interactions most likely are responsible for connectivity to various functions in most cases.…”

mentioning

confidence: 99%

Structure of a Construct of a Human Poly(C)-binding Protein Containing the First and Second KH Domains Reveals Insights into Its Regulatory Mechanisms

Fenn

Tjhen

et al. 2008

Journal of Biological Chemistry

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Poly(C)-binding proteins (PCBPs) 2 are KH (hnRNP-K-homology) domain-containing proteins that specifically recognize poly(C) D/RNA sequences (1, 2). There are five PCBPs in mammalian cells: PCBP1-4 and hnRNP K. Each PCBP contains three KH domains: two consecutive domains at the N terminus and a third domain at the C terminus; an intervening sequence of variable length is present between the second and third domains (Fig. 1A).PCBPs regulate gene expression at various levels, including transcription, mRNA processing, mRNA stabilization, and translation, among others. For example, specific binding of hnRNP K and PCBP1 to the single-stranded pyrimidine-rich promoter sequence of the human c-myc gene and mu-opioid receptor (MOR) gene, respectively, activates transcription (3, 4).Binding of PCBP1 or PCBP2 to cellular mRNAs harboring tandem poly(C) motifs within the 3Ј-UTRs stabilize these mRNAs, including ␣-globin, ␤-globin, collagen-␣1, tyrosine hydroxylase, erythropoietin, rennin, and hTERT mRNAs (5-13). In the case of ␣-globin mRNA, it was established that the stoichiometry of the RNA-protein complex (the ␣-complex) is 1:1, and a minimum RNA sequence of 20-nt (5Ј-CCCAACGGGCCCUCCUCCCC-3Ј) is necessary and sufficient for forming the complex (14).Interaction of two PCBPs, hnRNP K, and PCBP1/2, with a multiply tandem C-rich sequence (differentiation control element, DICE.) within the 3Ј-UTR of 15-lipoxygenase (LOX) mRNA leads to translational silencing of the mRNA in erythroid precursor cells (15-17). DICE contains 10 gapless C-rich repeats. The sequence for one repeat is 5Ј-CCCCACCCUCU-UCCCCAAG-3Ј. A minimum of two repeats is required for efficient translational suppression.PCBPs can also activate translation of cellular mRNAs. For example, binding of PCBP1 to an 18-nt C-rich sequence (5Ј-CUCCAUUCCCACUCCCU-3Ј) within the 5Ј-UTR of folate receptor mRNA up-regulates its translation (18). Binding of PCBP1/2 to the acute box cis-element in human heavy ferritin mRNA 5Ј-UTR also enhances translation (19).Besides cellular mRNAs, PCBPs also participate in regulating critical viral RNA functions. Binding of PCBP1/2 to two cisacting C-rich sequence-containing RNA elements within the 5Ј-UTR of poliovirus mRNA (also the genomic RNA) is critical for regulation of cap-independent translation and replication of the viral RNA (20 -24).The mechanistic details are not well understood for any of the PCBP functions. What emerges as a common feature is the binding of PCBPs to C-rich sequence motifs (often present in tandem) of the target D/RNAs. The molecular basis of PCBP KH domains-D/RNA interactions has been revealed by a number of crystal structures of individual KH1 or KH3 domain from PCBPs in complex with C-rich D/RNA sequences (25-28). However, there are no structures with KH2.Little is known about the events subsequent to any KH domain-D/RNA interaction. Pertinent to this point, there are no structures of PCBP constructs containing more than one KH * This work was supported, in whole or in part, by National Institutes of Health Gr...

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X-ray crystallographic and NMR studies of protein–protein and protein–nucleic acid interactions involving the KH domains from human poly(C)-binding protein-2

Cited by 57 publications

References 33 publications

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

Spatial arrangement of an RNA zipcode identifies mRNAs under post-transcriptional control

Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform

Structure of a Construct of a Human Poly(C)-binding Protein Containing the First and Second KH Domains Reveals Insights into Its Regulatory Mechanisms

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