ABSTRACT
The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45γ [corrected], revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45γ [corrected] identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45γ [corrected] is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.
Subject(s)
Animals , Humans , Mice , Amino Acid Motifs , Apoptosis , Radiation Effects , CDC2 Protein Kinase , Cell Cycle , Cell Survival , Crystallography, X-Ray , Cyclin B , Metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Cyclin-Dependent Kinases , HeLa Cells , Intracellular Signaling Peptides and Proteins , Chemistry , Genetics , Metabolism , Mutagenesis, Site-Directed , Mutation, Missense , Proliferating Cell Nuclear Antigen , Metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Ultraviolet RaysABSTRACT
The CCR4-NOT complex is a highly conserved, multifunctional machinery controlling mRNA metabolism. Its components have been implicated in several aspects of mRNA and protein expression, including transcription initiation, elongation, mRNA degradation, ubiquitination, and protein modification. In this review, we will focus on the role of the CCR4-NOT complex in mRNA degradation. The complex contains two types of deadenylase enzymes, one belonging to the DEDD-type family and one belonging to the EEP-type family, which shorten the poly(A) tails of mRNA. We will review the present state of structure-function analyses into the CCR4-NOT deadenylases and summarize current understanding of their roles in mRNA degradation. We will also review structural and functional work on the Tob/BTG family of proteins, which are known to interact with the CCR4-NOT complex and which have been reported to suppress deadenylase activity in vitro.
Subject(s)
Animals , Humans , Multiprotein Complexes , Chemistry , Genetics , Metabolism , Protein Conformation , RNA, Messenger , Genetics , Metabolism , Transcription Factors , Chemistry , Genetics , MetabolismABSTRACT
Enterovirus 71 (EV71), one of the major causative agents for hand-foot-and-mouth disease (HFMD), has caused more than 100 deaths among Chinese children since March 2008. The EV71 genome encodes an RNAdependent RNA polymerase (RdRp), denoted 3D(pol), which is central for viral genome replication and is a key target for the discovery of specific antiviral therapeutics. Here we report the crystal structures of EV71 RdRp (3D(pol)) and in complex with substrate guanosine-5'-triphosphate and analog 5-bromouridine-5'-triphosphate best to 2.4 Å resolution. The structure of EV71 RdRp (3D(pol)) has a wider open thumb domain compared with the most closely related crystal structure of poliovirus RdRp. And the EV71 RdRp (3D(pol)) complex with GTP or Br-UTP bounded shows two distinct movements of the polymerase by substrate or analogue binding. The model of the complex with the template:primer derived by superimposition with foot-and-mouth disease virus (FMDV) 3D/RNA complex reveals the likely recognition and binding of template:primer RNA by the polymerase. These results together provide a molecular basis for EV71 RNA replication and reveal a potential target for anti-EV71 drug discovery.
Subject(s)
Child , Humans , Amino Acid Sequence , China , Epidemiology , Crystallography, X-Ray , Drug Discovery , Enterovirus A, Human , Chemistry , Hand, Foot and Mouth Disease , Drug Therapy , Epidemiology , Virology , Models, Molecular , Molecular Sequence Data , Molecular Targeted Therapy , Protein Conformation , Protein Folding , RNA-Dependent RNA Polymerase , Chemistry , Genetics , Metabolism , Sequence Alignment , Substrate SpecificityABSTRACT
Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP(i)-generated and ATP-free enzyme reactions.
Subject(s)
Humans , Anti-HIV Agents , Pharmacology , Colorimetry , Diphosphates , Metabolism , Drug Evaluation, Preclinical , HIV , HIV Reverse Transcriptase , Metabolism , In Vitro Techniques , Nevirapine , Pharmacology , Reverse Transcriptase Inhibitors , Pharmacology , Sequence Analysis, DNA , Thymine Nucleotides , MetabolismABSTRACT
During severe acute respiratory syndrome coronavirus (SARS-CoV) infection, the activity of the replication/transcription complexes (RTC) quickly peaks at 6 hours post infection (h.p.i) and then diminishes significantly in the late post-infection stages. This "down-up-down" regulation of RNA synthesis distinguishes different viral stages: primary translation, genome replication, and finally viron assembly. Regarding the nsp8 as the primase in RNA synthesis, we confirmed that the proteolysis product of the primase (nsp8) contains the globular domain (nsp8C), and indentified the resectioning site that is notably conserved in all the three groups of coronavirus. We subsequently crystallized the complex of SARS-CoV nsp8C and nsp7, and the 3-D structure of this domain revealed its capability to interfuse into the hexadecamer super-complex. This specific proteolysis may indicate one possible mechanism by which coronaviruses to switch from viral infection to genome replication and viral assembly stages.
Subject(s)
Humans , Amino Acid Sequence , Crystallography, X-Ray , DNA Primase , Chemistry , Genetics , Physiology , Isoenzymes , Chemistry , Genetics , Physiology , Molecular Sequence Data , Protein Structure, Secondary , RNA, Viral , Severe acute respiratory syndrome-related coronavirus , Chemistry , Genetics , Physiology , Sequence Alignment , Severe Acute Respiratory Syndrome , Virology , Virus ReplicationABSTRACT
The important and diverse regulatory roles of Ca(2+) in eukaryotes are conveyed by the EF-hand containing calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still poorly understood. In this study, we report the three-dimensional structure of the calcium-binding protein from Streptomyces coelicolor, named CabD, which shares low sequence homology with other known helix-loop-helix EF-hand proteins. The CabD structure should provide insights into the biological role of the prokaryotic calcium-binding proteins. The unusual structural features of CabD compared with prokaryotic EF-hand proteins and eukaryotic sarcoplasmic calcium-binding proteins, including the bending conformation of the first C-terminal α-helix, unpaired ligand-binding EF-hands and the lack of the extreme C-terminal loop region, suggest it may have a distinct and significant function in calcium-mediated bacterial physiological processes, and provide a structural basis for potential calcium-mediated regulatory roles in prokaryotes.