Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28

The pluripotency factor Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal cold-shock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7g and that its C-terminal domain contributes predominantly to the affinity of this interaction. We uncover remarkable similarities between this C-terminal domain and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within pre-let-7g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind the extended loop of pre-let-7g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3′-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2–Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIEα uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair

Effects Of Nucleoside Analog Incorporation On Dna Binding To The Dna Binding Domain Of The Gata-1 Erythroid Transcription Factor

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-β-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site

Multiple Src Homology 3 Binding to the Ubiquitin Ligase Itch Conserved Proline-Rich Region

Itch is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases involved in the control of inflammatory signaling pathways, several transcription factors, and sorting of surface receptors to the degradative pathway. In addition to these common domains, Itch also contains a conserved proline-rich region (PRR) allowing its interaction with Src homology 3 (SH3) domain-containing proteins. This region is composed of 20 amino acids and contains one consensus class I and three class II SH3-binding motifs. Several SH3 domain-containing partners have been shown to recognize the Itch PRR, but their binding properties have been poorly defined. Here we compare a subset of endocytic SH3 domain-containing proteins using bioluminescence resonance energy transfer, isothermal titration calorimetry, and pull-down assays. Results indicate that Endophilin is a high-affinity binding partner of Itch both <i>in vivo</i> and <i>in vitro</i>, with a calculated <i>K</i>_D placing this complex among the highest-affinity SH3 domain-mediated interactions reported to date. All of the SH3 domains tested here bind to Itch with a 1:1 stoichiometry, except for β-PIX that binds with a 2:1 stoichiometry. Together, these results indicate that Itch PRR is a versatile binding module that can accommodate several different SH3 domain-containing proteins but has a preference for Endophilin. Interestingly, the catalytic activity of Itch toward different SH3 domain-containing proteins was similar, except for β-PIX that was not readily ubiquitylated even though it could interact with an affinity comparable to those of other substrates tested

Metal-dependent Ser/Thr protein phosphatase PPM family: Evolution, structures, diseases and inhibitors

© 2020 Elsevier Inc. Protein phosphatases and kinases control multiple cellular events including proliferation, differentiation, and stress responses through regulating reversible protein phosphorylation, the most important post-translational modification. Members of metal-dependent protein phosphatase (PPM) family, also known as PP2C phosphatases, are Ser/Thr phosphatases that bind manganese/magnesium ions (Mn2+/Mg2+) in their active center and function as single subunit enzymes. In mammals, there are 20 isoforms of PPM phosphatases: PPM1A, PPM1B, PPM1D, PPM1E, PPM1F, PPM1G, PPM1H, PPM1J, PPM1K, PPM1L, PPM1M, PPM1N, ILKAP, PDP1, PDP2, PHLPP1, PHLPP2, PP2D1, PPTC7, and TAB1, whereas there are only 8 in yeast. Phylogenetic analysis of the DNA sequences of vertebrate PPM isoforms revealed that they can be divided into 12 different classes: PPM1A/PPM1B/PPM1N, PPM1D, PPM1E/PPM1F, PPM1G, PPM1H/PPM1J/PPM1M, PPM1K, PPM1L, ILKAP, PDP1/PDP2, PP2D1/PHLPP1/PHLPP2, TAB1, and PPTC7. PPM-family members have a conserved catalytic core region, which contains the metal-chelating residues. The different isoforms also have isoform specific regions within their catalytic core domain and terminal domains, and these regions may be involved in substrate recognition and/or functional regulation of the phosphatases. The twenty mammalian PPM phosphatases are involved in regulating diverse cellular functions, such as cell cycle control, cell differentiation, immune responses, and cell metabolism. Mutation, overexpression, or deletion of the PPM phosphatase gene results in abnormal cellular responses, which lead to various human diseases. This review focuses on the structures and biological functions of the PPM-phosphatase family and their associated diseases. The development of specific inhibitors against the PPM phosphatase family as a therapeutic strategy will also be discussed