The Live Journal Journal Club

My most recent paper.

Structural basis for specificity in the poxvirus topoisomerase

Perry K, Hwang Y, Bushman FD, Van Duyne GD.

University of Pennsylvania School of Medicine, Department of Biochemistry and Biophysics and Howard Hughes Medical Institute, Philadelphia, Pennsylvania 19104, USA.

Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. The smallpox virus codes for its own topoisomerase enzyme that differs from its cellular counterpart by requiring a specific DNA sequence for activation of catalysis. Here we present crystal structures of the smallpox virus topoisomerase enzyme bound both covalently and noncovalently to a specific DNA sequence. These structures reveal the basis for site-specific DNA recognition, and they explain how catalysis is likely activated by formation of a specific enzyme-DNA interface. Unexpectedly, the poxvirus enzyme uses a major groove binding alpha helix that is not present in the human enzyme to recognize part of the core recognition sequence and activate the enzyme for catalysis. The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections.

Structural Activation Pathways from Dynamic Olfactory Receptor-Odorant Interactions (1662K)
PC Lai, MS Singer, and CJ Crasto
doi:10.1093/chemse/bji070

Olfactory receptors (ORs) are Class A GPCRs that bind small molecules and when activated, send signals to the brain via the olfactory nerve. The perception of smell is generated by the different activation profiles of different OR repertoires. ORs comprise the majority of GPCR genes in mammalian genomes. ORs and their odor ligands have a graded many-to-many binding (and activation) relationship, so that a single odor may activate different receptors to varying degree and a single OR may be capable of being activated by different odor ligands. The best theoretical way to evaluate the relationship between an OR's sequence and its activating ligands is structure-function analysis. While mutational studies can be carried out in the lab, theoretically, the current way to do this is to use rational pharmacophore-determining computational methods.
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I figured since I just joined, I should throw a paper into the mix. And it's a physical chemistry topic, so hopefully this will serve as a small drop in both the chemistry and physics buckets. :)

O.F.A. Larsen, et al. Probing the structure of a rotaxane with two-dimensional infrared spectroscopy. (2005) Proc. Nat. Acad. Sci. 102 (32): 13378-13382. (This is an Open Access article, so it should be no problem to bring up without a subscription.)

An interlocked complex of a macrocycle and a linear molecule was used as a model for molecular devices (this combination of a cyclic molecule locked onto a linear molecule is termed a rotaxane, two interlocked cyclic molecules would be known as a catenane for instance) that could be built for a variety of purposes. Since vibrational normal modes are usually well-defined since they involve stretching of a particular bond, using 2D IR spectroscopy would allow for measuring the coupling between the molecular vibrations in the molecule or complex. In this experiment, the vibrational modes of the carbonyl (C=O) groups were used to extract out the conformation (distances and angular constraints) of the rotaxane assembly.

Any IR active bond will do, for example, the amide transitions could also be used profitably. As a supplement and further background, this paper (also from PNAS, older than six months so it's open for anyone) shows that the amide transitions were used to monitor conformational changes of the unfolding of ubiquitin.

Extension to other optical regimes has been explored. In a paper that came out this past year, studies of the electronic coupling and energy transfer in a photosynthetic protein were done in the visible light range (T. Brixner, et al. "Two-dimensional spectroscopy of electronic couplings in photosynthesis." (2005) Nature. 434: 625-628 (Link not given since Nature is a pain in the tail about open access in general...).

The advantages of exploring dynamics and function with high time resolution are incredible, although the sort of structure determination that can be done currently with NMR and EPR is probably some time off into the future. Although as my former advisor once said, molecules do things and we want to understand what they do - there's more to it than just how the atoms are arranged, after all. So that was more like a paper and then some additional information...

o O (Hopes there are still people reading this community....)

All my thanks to those of you who have posted articles. They have been excellent so far!

I'd love to see some of your non-biology type folks post something....perhaps a good chem or physics article?

Love and kisses,
Fenske Melissa

So perhaps these results are already well known... I think every self respecting biochemist and geneticist should give these a read out of historical relevance. They're also worth it for a chuckle about the kinds of assertions you could make 50 years ago. I present:

Molecular structure of Nucleic Acids
WATSON, J. D. & CRICK, F. H. C.
Nature 171, 737-738 (1953)
URL: http://www.nature.com/genomics/hum…
Annotated version (I recommend this one, actually)


Molecular Structure of Deoxypentose Nucleic Acids
Wilkins, M. H. F., Stokes, A. R., & Wilson, H. R.
Nature 171, 738-740 (1953)
URL: http://www.nature.com/nature/dna50…


Molecular Configuration in Sodium Thymonucleate
Franklin, R., and Gosling, R. G.
Nature 171, 740-741 (1953)
URL: http://www.nature.com/nature/dna50…



This site ( http://www.ba-education.demon.co.u…) has a good history of the discovery. These papers, 2 followups (all 5 in Nature), and the original paper showing that DNA is the molecule of inheretence (from the Journal of Medicinal Chemistry) are made available at Nature's 50 Years of DNA website (http://www.nature.com/nature/dna5…

Watson and Crick produced none of the data themselves; they took it all from Rosalind Franklin, mostly without her knowledge or consent. These three, and also Maurice Wilkins, worked in sister labs, so had a fair amount of contact with each other. To be fair, none of these scientists were nice people, this article implies that if they hadn't let their egos get in the way and had collaborated, they would have found the structure a year earlier. Instead, they all published competing papers in the April 1953 issue of Nature. Watson and Crick were awarded the 1962 Nobel Prize in Physiology or Medicine. Franklin might have been included in the prize, but she passed away in 1958, and the statutes of the Nobel Foundation state that awards shall not be made to the diseased.

Field: Biochemistry
Topic: DNA Rotation by Topoisomerase I

Proc Natl Acad Sci U S A. 2003 May 13;100(10):5640-5. Epub 2003 Apr 23.
DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein.

Carey JF, Schultz SJ, Sisson L, Fazzio TG, Champoux JJ.

Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195, USA.

In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3' end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the "lips" region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp.

versus

Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):13767-72. Epub 2003 Oct 29.

Locking the DNA topoisomerase I protein clamp inhibits DNA rotation and induces cell lethality.

Woo MH, Losasso C, Guo H, Pattarello L, Benedetti P, Bjornsti MA.

Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.

Eukaryotic DNA topoisomerase I (Top1) is a monomeric protein clamp that functions in DNA replication, transcription, and recombination. Opposable "lip" domains form a salt bridge to complete Top1 protein clamping of duplex DNA. Changes in DNA topology are catalyzed by the formation of a transient phosphotyrosyl linkage between the active-site Tyr-723 and a single DNA strand. Substantial protein domain movements are required for DNA binding, whereas the tight packing of DNA within the covalent Top1-DNA complex necessitates some DNA distortion to allow rotation. To investigate the effects of Top1-clamp closure on enzyme catalysis, molecular modeling was used to design a disulfide bond between residues Gly-365 and Ser-534, to crosslink protein loops more proximal to the active-site tyrosine than the protein loops held by the Lys-369-Glu-497 salt bridge. In reducing environments, Top1-Clamp was catalytically active. However, contrary to crosslinking the salt-bridge loops [Carey, J. F., Schultz, S. J., Sission, L., Fazzio, T. G. & Champoux, J. J. (2003) Proc. Natl. Acad. Sci. USA 100, 5640-5645], crosslinking the active-site proximal loops inhibited DNA rotation. Apparently, subtle alterations in Top1 clamp flexibility impact enzyme catalysis in vitro. Yet, the catalytically active Top1-Clamp was cytotoxic, even in the reducing environment of yeast cells. Remarkably, a shift in redox potential in glr1Delta cells converted the catalytically inactive Top1Y723F mutant clamp into a cellular toxin, which failed to induce an S-phase terminal phenotype. This cytotoxic mechanism is distinct from that of camptothecin chemotherapeutics, which stabilize covalent Top1-DNA complexes, and it suggests that the development of novel therapeutics that promote Top1-clamp closure is possible.

Both of these articles should be available to everyone.

In summary, there is a prevailing hypothesis that Type IB topoisomerases (of which Human Topo I is a family member), relax DNA through controlled rotation or friction between the protein and the DNA. The enzyme forms a C-shaped clamp around the DNA, nicks a single strand of the duplex and then allows the uncut strand to rotate around the nick. The method by which this rotation occurs is still unknown. Of these two papers, one group claims that DNA can rotate while the enzyme is closed around it and the other groups claims that DNA cannot rotate while the enzyme is closed around it. Which paper do you believe? Which group does the more believeable experiment? I personally think that Carey et al do not perform the proper negative controls. Do you agree with me or disagree? Why?

Finally, if one the conclusions of one of these papers is wrong, what happens? Do the authors eventually publish a retraction? Or, does it simply become a dinosaur, unmentioned/unreferenced in future papers?

I found this article in reading on GRB in a current issue of astro-ph.

Kobayashi and Zhang; Early Optical Afterglows from wind-type gamma-ray Bursts from the Astrophysical journal, 597:455-458, 2003 Nov 1

I don't think in general APJ is free so I used the above link to arxiv because I think in this format it is free for everyone.

This article I thought did a good job at introducing the wind type grb as a new scenario for the light curves we see from GRB's. They not only provide a detailed analytic case but then they do a few case studies, although I wish they would include more figures for the case studies for example light curve.

The paper spends some depth on the topic of angular time delay effects for the wind model and it seems like this effect changes the light curve dramatically from the ISM case to the wind model, but it doesn't say why angular time delay effect doesn't happen in the ISM case. I also can't find a paper that mentions this. So why is the angular time delay effect negligible or not present in the ISM case?

Field: Biochemistry
Authors: S. D. Hatfield1, H. R. Shcherbata, K. A. Fischer, K. Nakahara, R. W. Carthew and H. Ruohola-Baker
Citation: Nature. 2005 Jun 16;435(7044):974-8. Epub 2005 Jun 8.
Links: Nature. Pubmed
Abstract

One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

Summary
This was one of my favorite papers this year. The micro RNA (miRNA) (also RNA interference (RNAi)) pathway is a biochemical system discovered only about 10 years ago (brand new in biochem terms!). They only discovered and crystalized the second key protein in this system last year, and it's a hot field a research seeing what all micro RNA's are involved in.

In short, miRNAs are short (~23 base pairs) sequences complimentary to RNA transcriptions of some genes. These miRNAs recruit a large protein complex called RISC, and RISC holds onto the complimentary RNAs and prevent their translation into proteins. Thus miRNAs are a mechanism of gene regulation. You can read a nice review here.

Most cells in your body have slowed or stopped dividing, with one notable exception of stem cells. These guys keep going and going and must have some mechanism for defeating the normal inhibition of division. Surprisingly, the miRNA pathway presents an elegant solution.

Questions
Will this system be implicated in other dividing cells?
Do some cancers have mutations in the RNAi pathway?
Where will RNAi turn up next?

Update
My friend reminds me that there was a significant problem in this paper. Sadly he doesn't remember what it is either, so we don't know how significant. Bonus points for anyone who can find it (I suppose I should reread the thing now and see if I can find it too).

Allow me to submit the first article for discussion...I found this article to be of interest as someone who works on the effects of airborne pollutants on smokers...

Matt X. G. Shao et al. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor--converting enzyme in human airway epithelial (NCI-H292) cells. Am J Physiol Lung Cell Mol Physiol 287: L420-L427, 2004.

The article is free to anyone who want to access it.

I think that the article does a fairly good job at discussing the scope of the problem.

Reading this article made me wonder about some of the following questions:

1)The authors contend that reactive oxygen species (ROS) are responsible for TACE activation, however, administration of antioxidants (both oral and topical) to individuals with COPD and asthma has had less than stellar results. Additionally, smokers tend to upregulate expression of things like GSH and sequester more uric acid and vitamin e. I wonder if there are other things that might activate TACE and, if their experiments were carried out in an antioxidant supplied environment, if they would have seen the same levels of activation. Still...their results were compelling...

2)The last sentence of their article reads "The discovery that cigarette smoke-induced mucin overproduction is mediated by TACE activation suggests new therapies for COPD." What would treatment with TGF-a scavengers or TACE inactivators show in vivo. Could they prevent an animal model from developing COPD after long term cigarette exposure? What about an EGFR blocker? Would that also prevent the airway remodeling seen in these diseases?

3)What is the long term effect of decreasing mucin production? Mucin is important in the protective layer of the lung. Would the trade off for less mucin be a more necrotic lung?