The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications

2015 ◽  
Vol 43 (5) ◽  
pp. 819-824 ◽  
Author(s):  
Marie Valerio-Lepiniec ◽  
Agathe Urvoas ◽  
Anne Chevrel ◽  
Asma Guellouz ◽  
Yann Ferrandez ◽  
...  
Keyword(s):  
Dissociation Constants ◽  
Elongation Factor ◽  
Protein Targets ◽  
Artificial Proteins ◽  
X Ray Crystallography ◽  
New Family ◽  
Cell Compartments ◽  
Repeat Proteins ◽  
Elongation Factor 3 ◽  
Repeated Motifs

We have designed a new family of artificial proteins, named αRep, based on HEAT (acronym for Huntingtin, elongation factor 3 (EF3), protein pphosphatase 2A (PP2A), yeast kinase Tor1) repeat proteins containing an α-helical repeated motif. The sequence of the repeated motifs, first identified in a thermostable archae protein was optimized using a consensus design strategy and used for the construction of a library of artificial proteins. All proteins from this library share the same general fold but differ both in the number of repeats and in five highly randomized amino acid positions within each repeat. The randomized side chains altogether provide a hypervariable surface on αRep variants. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins. αRep binders with high affinity for various protein targets were selected by phage display. Low micromolar to nanomolar dissociation constants between partners were measured and the structures of several complexes (specific αRep/protein target) were solved by X-ray crystallography. Using GFP as a model target, it was demonstrated that αReps can be used as bait in pull-down experiments. αReps can be expressed in eukaryotic cells and specifically interact with their target addressed to different cell compartments.

scholarly journals Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening

2018 ◽  
Vol 19 (11) ◽  
pp. 3664 ◽  
Author(s):  
Ali Munawar ◽  
Steven Beelen ◽  
Ahmad Munawar ◽  
Eveline Lescrinier ◽  
Sergei Strelkov
Keyword(s):  
Zika Virus ◽  
Dissociation Constants ◽  
Global Public Health ◽  
Human Pathogens ◽  
Allosteric Site ◽  
Replication Complex ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
Atomic Precision

The flavivirus family contains several important human pathogens, such as Zika virus (ZIKV), dengue, West Nile, and Yellow Fever viruses, that collectively lead to a large, global disease burden. Currently, there are no approved medicines that can target these viruses. The sudden outbreak of ZIKV infections in 2015–2016 posed a serious threat to global public health. While the epidemic has receded, persistent reservoirs of ZIKV infection can cause reemergence. Here, we have used X-ray crystallography-based screening to discover two novel sites on ZIKV NS3 helicase that can bind drug-like fragments. Both sites are structurally conserved in other flaviviruses, and mechanistically significant. The binding poses of four fragments, two for each of the binding sites, were characterized at atomic precision. Site A is a surface pocket on the NS3 helicase that is vital to its interaction with NS5 polymerase and formation of the flaviviral replication complex. Site B corresponds to a flexible, yet highly conserved, allosteric site at the intersection of the three NS3 helicase domains. Saturation transfer difference nuclear magnetic resonance (NMR) experiments were additionally used to evaluate the binding strength of the fragments, revealing dissociation constants (KD) in the lower mM range. We conclude that the NS3 helicase of flaviviruses is a viable drug target. The data obtained open opportunities towards structure-based design of first-in-class anti-ZIKV compounds, as well as pan-flaviviral therapeutics.

scholarly journals Detection of secondary binding sites in proteins using fragment screening

2015 ◽  
Vol 112 (52) ◽  
pp. 15910-15915 ◽  
Author(s):  
R. Frederick Ludlow ◽  
Marcel L. Verdonk ◽  
Harpreet K. Saini ◽  
Ian J. Tickle ◽  
Harren Jhoti
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Cellular Functions ◽  
Modulate Function ◽  
Protein Targets ◽  
Biologically Relevant ◽  
X Ray Crystallography ◽  
Fragment Screening ◽  
High Conservation ◽  
Partner Proteins

Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

scholarly journals Recent Progress in RXLR Effector Research

2015 ◽  
Vol 28 (10) ◽  
pp. 1063-1072 ◽  
Author(s):  
Ryan G. Anderson ◽  
Devdutta Deb ◽  
Kevin Fedkenheuer ◽  
John M. McDowell
Keyword(s):  
Comparative Genomics ◽  
Effector Proteins ◽  
Host Protein ◽  
Protein Targets ◽  
Major Function ◽  
Functional Studies ◽  
Rxlr Effectors ◽  
Cell Compartments ◽  
Rxlr Effector ◽  
Bacteria And Fungi

Some of the most devastating oomycete pathogens deploy effector proteins, with the signature amino acid motif RXLR, that enter plant cells to promote virulence. Research on the function and evolution of RXLR effectors has been very active over the decade that has transpired since their discovery. Comparative genomics indicate that RXLR genes play a major role in virulence for Phytophthora and downy mildew species. Importantly, gene-for-gene resistance against these oomycete lineages is based on recognition of RXLR proteins. Comparative genomics have revealed several mechanisms through which this resistance can be broken, most notably involving epigenetic control of RXLR gene expression. Structural studies have revealed a core fold that is present in the majority of RXLR proteins, providing a foundation for detailed mechanistic understanding of virulence and avirulence functions. Finally, functional studies have demonstrated that suppression of host immunity is a major function for RXLR proteins. Host protein targets are being identified in a variety of plant cell compartments. Some targets comprise hubs that are also manipulated by bacteria and fungi, thereby revealing key points of vulnerability in the plant immune network.

Polyamino Acids That Inhibit the Interaction of Yeast Translational Elongation Factor-3 (EF-3) with Ribosomes

1994 ◽  
Vol 115 (5) ◽  
pp. 820-824 ◽  
Author(s):  
Masahiro Uritani ◽  
Kyoko Nakano ◽  
Yuhko Aoki ◽  
Hisao Shimada ◽  
Mikio Arisawa
Keyword(s):  
Elongation Factor ◽  
Elongation Factor 3

scholarly journals Using iterative fragment assembly and progressive sequence truncation to facilitate phasing and crystal structure determination of distantly related proteins

2016 ◽  
Vol 72 (5) ◽  
pp. 616-628 ◽  
Author(s):  
Yan Wang ◽  
Jouko Virtanen ◽  
Zhidong Xue ◽  
John J. G. Tesmer ◽  
Yang Zhang
Keyword(s):  
Success Rate ◽  
Structure Determination ◽  
Protein Targets ◽  
Search Models ◽  
X Ray Crystallography ◽  
Structure Genomics ◽  
Related Proteins ◽  
Structure Assembly ◽  
Sequence Truncation

Molecular replacement (MR) often requires templates with high homology to solve the phase problem in X-ray crystallography.I-TASSER-MRhas been developed to test whether the success rate for structure determination of distant-homology proteins could be improved by a combination of iterative fragmental structure-assembly simulations with progressive sequence truncation designed to trim regions with high variation. The pipeline was tested on two independent protein sets consisting of 61 proteins from CASP8 and 100 high-resolution proteins from the PDB. After excluding homologous templates,I-TASSERgenerated full-length models with an average TM-score of 0.773, which is 12% higher than the best threading templates. Using these as search models,I-TASSER-MRfound correct MR solutions for 95 of 161 targets as judged by having a TFZ of >8 or with the final structure closer to the native than the initial search models. The success rate was 16% higher than when using the best threading templates.I-TASSER-MRwas also applied to 14 protein targets from structure genomics centers. Seven of these were successfully solved byI-TASSER-MR. These results confirm that advanced structure assembly and progressive structural editing can significantly improve the success rate of MR for targets with distant homology to proteins of known structure.

Sign in / Sign up

Export Citation Format

Share Document