scholarly journals Structure of substrate-bound SMG1-8-9 kinase complex reveals molecular basis for phosphorylation specificity

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lukas M Langer ◽  
Yair Gat ◽  
Fabien Bonneau ◽  
Elena Conti
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Phosphorylation Site ◽  
Hydrophobic Residue ◽  
Cellular Processes ◽  
Cryo Electron Microscopy ◽  
Biochemical Assays ◽  
Rna Quality Control ◽  
Nonsense Mediated Mrna Decay ◽  
Site Recognition

PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.

scholarly journals Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Timothy P. Newing ◽  
Aaron J. Oakley ◽  
Michael Miller ◽  
Catherine J. Dawson ◽  
Simon H. J. Brown ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Conformational Changes ◽  
Active Site ◽  
Molecular Basis ◽  
Replication Machinery ◽  
Gram Positive Bacteria ◽  
Cryo Electron Microscopy ◽  
Large Conformational Change ◽  
Transcription Complexes ◽  
Primary Channel

AbstractIn bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

scholarly journals 3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Edward J Brignole ◽  
Kuang-Lei Tsai ◽  
Johnathan Chittuluru ◽  
Haoran Li ◽  
Yimon Aye ◽  
...  
Keyword(s):  
Active Site ◽  
Molecular Basis ◽  
Α Subunit ◽  
Cryo Electron Microscopy ◽  
Ribonucleotide Reductases ◽  
Dna Replication And Repair ◽  
Radical Transfer ◽  
Allosteric Regulators ◽  
Insight Into ◽  
Resolution Structure

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and β2. Together, these structures support a model for RNR inhibition in which β2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.

scholarly journals Cryo-EM reconstructions of inhibitor-bound SMG1 kinase reveal an autoinhibitory state dependent on SMG8

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lukas M Langer ◽  
Fabien Bonneau ◽  
Yair Gat ◽  
Elena Conti
Keyword(s):  
Small Molecule ◽  
Molecular Basis ◽  
Biochemical Analysis ◽  
Kinase Activity ◽  
Regulatory Mechanisms ◽  
Cryo Electron Microscopy ◽  
State Dependent ◽  
Molecule Inhibitor ◽  
Nonsense Mediated Mrna Decay ◽  
Complex Regulatory Network

The PI3K-related kinase (PIKK) SMG1 monitors progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 is impaired by small molecule inhibitors, is reduced by the SMG1 interactors SMG8 and SMG9, and is downregulated by the so-called SMG1 insertion domain. However, the molecular basis for this complex regulatory network has remained elusive. Here, we present cryo-electron microscopy reconstructions of human SMG1-9 and SMG1-8-9 complexes bound to either a SMG1 inhibitor or a non-hydrolyzable ATP analogue at overall resolutions ranging from 2.8 to 3.6 Å. These structures reveal the basis with which a small molecule inhibitor preferentially targets SMG1 over other PIKKs. By comparison with our previously reported substrate-bound structure (Langer et al. 2020), we show that the SMG1 insertion domain can exert an autoinhibitory function by directly blocking the substrate binding path as well as overall access to the SMG1 kinase active site. Together with biochemical analysis, our data indicate that SMG1 autoinhibition is stabilized by the presence of SMG8. Our results explain the specific inhibition of SMG1 by an ATP-competitive small molecule, provide insights into regulation of its kinase activity within the NMD pathway, and expand the understanding of PIKK regulatory mechanisms in general.

scholarly journals Cryo-EM reconstructions of inhibitor-bound SMG1 kinase reveal an autoinhibitory state dependent on SMG8

2021 ◽  
Author(s):  
Lukas M Langer ◽  
Fabien Bonneau ◽  
Yair Gat ◽  
Elena Conti
Keyword(s):  
Small Molecule ◽  
Molecular Basis ◽  
Biochemical Analysis ◽  
Kinase Activity ◽  
Regulatory Mechanisms ◽  
Cryo Electron Microscopy ◽  
State Dependent ◽  
Molecule Inhibitor ◽  
Nonsense Mediated Mrna Decay ◽  
Complex Regulatory Network

The PI3K-related kinase (PIKK) SMG1 monitors progression of metazoan nonsense-mediated mRNA decay (NMD) by phosphorylating the RNA helicase UPF1. Previous work has shown that the activity of SMG1 is impaired by small molecule inhibitors, is reduced by the SMG1 interactors SMG8 and SMG9, and is downregulated by the so-called SMG1 insertion domain. However, the molecular basis for this complex regulatory network has remained elusive. Here, we present cryo-electron microscopy reconstructions of human SMG1-9 and SMG1-8-9 complexes bound to either a SMG1 inhibitor or a non-hydrolyzable ATP analogue at overall resolutions ranging from 2.8 to 3.6 Å. These structures reveal the basis with which a small molecule inhibitor preferentially targets SMG1 over other PIKKs. By comparison with our previously reported substrate-bound structure (Langer et al. 2020), we show that the SMG1 insertion domain can exert an autoinhibitory function by directly blocking the substrate binding path as well as overall access to the SMG1 kinase active site. Together with biochemical analysis, our data indicate that SMG1 autoinhibition is stabilized by the presence of SMG8. Our results explain the specific inhibition of SMG1 by an ATP-competitive small molecule, provide insights into regulation of its kinase activity within the NMD pathway, and expand the understanding of PIKK regulatory mechanisms in general.

scholarly journals Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB.

1984 ◽  
Vol 259 (22) ◽  
pp. 14184-14189
Author(s):  
J P Albanesi ◽  
H Fujisaki ◽  
E D Korn
Keyword(s):  
Active Site ◽  
Phosphorylation Site

scholarly journals Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yi Zhang ◽  
Kyle Brown ◽  
Yucong Yu ◽  
Ziad Ibrahim ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cell Nucleus ◽  
Active Site ◽  
Histone Acetyltransferase ◽  
Histone H3 ◽  
Catalytic Core ◽  
Cellular Processes ◽  
Storage Pool ◽  
Core Components ◽  
Acetyltransferase P300

AbstractThe transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

scholarly journals High-resolution structures of inhibitor complexes of human indoleamine 2,3-dioxygenase 1 in a new crystal form

2018 ◽  
Vol 74 (11) ◽  
pp. 717-724 ◽  
Author(s):  
Shukun Luo ◽  
Ke Xu ◽  
Shaoyun Xiang ◽  
Jie Chen ◽  
Chunyun Chen ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Resolution ◽  
Crystal Structures ◽  
Active Site ◽  
Crystal Form ◽  
Crystal Forms ◽  
Cellular Processes ◽  
Potential Target ◽  
Indoleamine 2,3 Dioxygenase

Human indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme with important roles in many cellular processes and is a potential target for drug discovery against cancer and other diseases. Crystal structures of IDO1 in complex with various inhibitors have been reported. Many of these crystals belong to the same crystal form and most of the reported structures have resolutions in the range 3.2–2.3 Å. Here, three new crystal forms of human IDO1 obtained by introducing a surface mutation, K116A/K117A, distant from the active site are reported. One of these crystal forms diffracted to 1.5 Å resolution and can be readily used for soaking experiments to determine high-resolution structures of IDO1 in complex with the substrate tryptophan or inhibitors that coordinate the heme. In addition, this mutant was used to produce crystals of a complex with an inhibitor that targets the apo form of the enzyme under the same conditions; the structure of this complex was determined at 1.7 Å resolution. Overall, this mutant represents a robust platform for determining the structures of inhibitor and substrate complexes of IDO1 at high resolution.

scholarly journals Mycoplasmas and Their Antibiotic Resistance: The Problems and Prospects in Controlling Infections

Acta Naturae ◽  
2016 ◽  
Vol 8 (2) ◽  
pp. 24-34 ◽  
Author(s):  
O. A. Chernova ◽  
E. S. Medvedeva ◽  
A. A. Mouzykantov ◽  
N. B. Baranova ◽  
V. M. Chernov
Keyword(s):  
Antibiotic Resistance ◽  
Molecular Basis ◽  
Rapid Development ◽  
Integrated Approach ◽  
Stress Factors ◽  
Antimicrobial Drugs ◽  
Cellular Processes ◽  
Complex Processes ◽  
Development Of Resistance ◽  
Higher Eukaryotes

The present review discusses the problem of controlling mycoplasmas (class Mollicutes), the smallest of self-replicating prokaryotes, parasites of higher eukaryotes, and main contaminants of cell cultures and vaccines. Possible mechanisms for the rapid development of resistance to antimicrobial drugs in mycoplasmas have been analyzed. Omics technologies provide new opportunities for investigating the molecular basis of bacterial adaptation to stress factors and identifying resistomes, the total of all genes and their products contributing to antibiotic resistance in microbes. The data obtained using an integrated approach with post-genomics methods show that antibiotic resistance may be caused by more complex processes than has been believed heretofore. The development of antibiotic resistance in mycoplasmas is associated with essential changes in the genome, proteome, and secretome profiles, which involve many genes and proteins related to fundamental cellular processes and virulence.

scholarly journals A UPF1 variant drives conditional remodeling of nonsense-mediated mRNA decay

2021 ◽  
Author(s):  
Sarah E. Fritz ◽  
Soumya Ranganathan ◽  
J. Robert Hogg
Keyword(s):  
Gene Expression ◽  
Mrna Decay ◽  
Gene Expression Regulation ◽  
Translation Termination ◽  
Translational Repression ◽  
The Core ◽  
Human Genes ◽  
Rna Quality Control ◽  
Nonsense Mediated Mrna Decay

AbstractThe nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Due to the major role of NMD in RNA quality control and gene expression regulation, it is important to understand how the pathway responds to changing cellular conditions. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. UPF1LL associates more stably with potential NMD target mRNAs than the major UPF1SL isoform, expanding the scope of NMD to include many transcripts normally immune to the pathway. Unexpectedly, the enhanced persistence of UPF1LL on mRNAs supports induction of NMD in response to rare translation termination events. Thus, while canonical NMD is abolished by translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress.

scholarly journals Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C1211-C1211
Author(s):  
Joseph Ng ◽  
Ronny Hughes ◽  
Michelle Morris ◽  
Leighton Coates ◽  
Matthew Blakeley ◽  
...  
Keyword(s):  
High Resolution ◽  
Active Site ◽  
Inorganic Pyrophosphatase ◽  
Inorganic Pyrophosphate ◽  
X Ray ◽  
X Ray Crystallography ◽  
Cellular Processes ◽  
Crystallographic Structures ◽  
Hydrolysis Of ◽  
Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

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