scholarly journals Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode

2017 ◽  
Vol 73 (4) ◽  
pp. 294-315 ◽  
Author(s):  
Kimberly A. Stanek ◽  
Jennifer Patterson-West ◽  
Peter S. Randolph ◽  
Cameron Mura
Keyword(s):  
Rna Binding ◽  
Binding Pocket ◽  
Binding Mode ◽  
Evolutionary Conservation ◽  
Aquifex Aeolicus ◽  
Binding Properties ◽  
Bacterial Phyla ◽  
Mrna Targets ◽  
Small Regulatory Rnas ◽  
And Function

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophileAquifex aeolicus(Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore,AaeHfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures ofAaeHfq were determined in space groupsP1 andP6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6RNA reveals that the outer rim of theAaeHfq hexamer features a well defined binding pocket that is selective for uracil. ThisAaeHfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

scholarly journals Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: Conservation of the lateral RNA-binding mode

2016 ◽  
Author(s):  
Kimberly A Stanek ◽  
Jennifer P West ◽  
Peter S Randolph ◽  
Cameron Mura
Keyword(s):  
Rna Binding ◽  
Binding Pocket ◽  
Binding Mode ◽  
Evolutionary Conservation ◽  
Thermophilic Bacterium ◽  
Aquifex Aeolicus ◽  
Binding Properties ◽  
Bacterial Phyla ◽  
Mrna Targets ◽  
Small Regulatory Rnas

SynopsisThe structure of an Hfq homolog from the deep-branching thermophilic bacterium Aquifex aeolicus, determined to 1.5-Å resolution both in apo form and bound to a uridine-rich RNA, reveals a conserved, pre-organized RNA-binding pocket on the lateral rim of the Hfq hexamer.AbstractThe host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNA) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings, with at least two distinct surfaces that bind RNA. Recently, another binding site—dubbed the ‘lateral rim’—has been implicated in sRNA•mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structures and functions of Hfq from basal bacterial lineages such as the Aquificae. Thus, we have cloned, overexpressed, purified, crystallized, and biochemically characterized Aae Hfq. We have determined the structures of Aae Hfq in space-groups P1 and P6, both to 1.5 Å resolution, and we have discovered nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well-defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

scholarly journals Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets

PLoS Biology ◽  
2015 ◽  
Vol 13 (11) ◽  
pp. e1002307 ◽  
Author(s):  
Gregory J. Hogan ◽  
Patrick O. Brown ◽  
Daniel Herschlag
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Evolutionary Conservation ◽  
Mrna Targets

scholarly journals Elucidating the tunability of binding behavior for the MERS-CoV macro domain with NAD metabolites

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Meng-Hsuan Lin ◽  
Chao-Cheng Cho ◽  
Yi-Chih Chiu ◽  
Chia-Yu Chien ◽  
Yi-Ping Huang ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Binding Mode ◽  
Biological Functions ◽  
Binding Properties ◽  
Binding Ability ◽  
Nad Metabolism ◽  
Ligand Selectivity ◽  
Physiological Temperature ◽  
Binding Behavior ◽  
Macro Domain

AbstractThe macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.

scholarly journals A second PI(4,5)P2 binding site determines PI(4,5)P2 sensitivity of the tubby domain

2020 ◽  
Author(s):  
Veronika Thallmair ◽  
Lea Schultz ◽  
Siewert J. Marrink ◽  
Dominik Oliver ◽  
Sebastian Thallmair
Keyword(s):  
Binding Site ◽  
Md Simulations ◽  
Binding Pocket ◽  
Binding Mode ◽  
Phosphatidyl Serine ◽  
Coarse Grained ◽  
Membrane Association ◽  
Membrane Interface ◽  
Binding Properties ◽  
Protein Membrane

ABSTRACTPhosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic.Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico.Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.

scholarly journals Brothers in Arms: Structure, Assembly and Function of Arenaviridae Nucleoprotein

Viruses ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 772 ◽  
Author(s):  
Nicolas Papageorgiou ◽  
Maria Spiliopoulou ◽  
Thi-Hong Van Nguyen ◽  
Afroditi Vaitsopoulou ◽  
Elsie Yekwa Laban ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Rna Binding ◽  
Binding Mode ◽  
Structural Motif ◽  
Emerging Pathogens ◽  
Functional Implications ◽  
And Function ◽  
Structure Assembly ◽  
Domain Exon

Arenaviridae is a family of viruses harbouring important emerging pathogens belonging to the Bunyavirales order. Like in other segmented negative strand RNA viruses, the nucleoprotein (NP) is a major actor of the viral life cycle being both (i) the necessary co-factor of the polymerase present in the L protein, and (ii) the last line of defence of the viral genome (vRNA) by physically hiding its presence in the cytoplasm. The NP is also one of the major players interfering with the immune system. Several structural studies of NP have shown that it features two domains: a globular RNA binding domain (NP-core) in its N-terminal and an exonuclease domain (ExoN) in its C-terminal. Further studies have observed that significant conformational changes are necessary for RNA encapsidation. In this review we revisited the most recent structural and functional data available on Arenaviridae NP, compared to other Bunyavirales nucleoproteins and explored the structural and functional implications. We review the variety of structural motif extensions involved in NP–NP binding mode. We also evaluate the major functional implications of NP interactome and the role of ExoN, thus making the NP a target of choice for future vaccine and antiviral therapy.

scholarly journals Structure and Function of Flavivirus NS5 Methyltransferase

2007 ◽  
Vol 81 (8) ◽  
pp. 3891-3903 ◽  
Author(s):  
Yangsheng Zhou ◽  
Debashish Ray ◽  
Yiwei Zhao ◽  
Hongping Dong ◽  
Suping Ren ◽  
...  
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Binding Pocket ◽  
Wild Type ◽  
Terminal Portion ◽  
Novel Target ◽  
And Function ◽  
Methylation Activity ◽  
Single Binding Site ◽  
Positively Charged

ABSTRACT The plus-strand RNA genome of flavivirus contains a 5′ terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA → m7GpppA → m7GpppAm. The 2′-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 Å resolution showed a single binding site for S-adenosyl-l-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2′-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2′-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

scholarly journals Spanning the gap: unraveling RSC dynamics in vivo

2021 ◽  
Author(s):  
Heinz Neumann ◽  
Bryan J. Wilkins
Keyword(s):  
Cell Cycle ◽  
Posttranslational Modifications ◽  
Transcriptional Activation ◽  
Binding Mode ◽  
Binding Modes ◽  
Binding Domains ◽  
Binding Interface ◽  
The Many ◽  
And Function

AbstractMultiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

scholarly journals Exploring aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction: an in silico study

2021 ◽  
Author(s):  
Chiara Luise ◽  
Dina Robaa ◽  
Wolfgang Sippl
Keyword(s):  
Molecular Dynamic ◽  
In Silico ◽  
Binding Pocket ◽  
Binding Mode ◽  
Molecular Dynamic Simulations ◽  
Flexible Docking ◽  
Dynamic Simulations ◽  
Binding Mode Prediction ◽  
Aromatic Cage ◽  
The Right

AbstractSome of the main challenges faced in drug discovery are pocket flexibility and binding mode prediction. In this work, we explored the aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction by means of in silico approaches. We first investigated the Spindlin1 aromatic cage plasticity by analyzing the available crystal structures and through molecular dynamic simulations. Then we assessed the ability of rigid docking and flexible docking to rightly reproduce the binding mode of a known ligand into Spindlin1, as an example of a reader protein displaying flexibility in the binding pocket. The ability of induced fit docking was further probed to test if the right ligand binding mode could be obtained through flexible docking regardless of the initial protein conformation. Finally, the stability of generated docking poses was verified by molecular dynamic simulations. Accurate binding mode prediction was obtained showing that the herein reported approach is a highly promising combination of in silico methods able to rightly predict the binding mode of small molecule ligands in flexible binding pockets, such as those observed in some reader proteins.

Sign in / Sign up

Export Citation Format

Share Document