Base flipping: still some unanswered questions?

It is now 20 years since the original paper by Xiaodong Cheng, Rich Roberts and colleagues was published: it remains a landmark in our understanding of the way in which nucleic acid modifying enzymes access target bases. Until that time, target bases appeared to be stably satisfied by Watson and Crick base pairing. The subsequent analysis of C5 MTase open reading frames by many labs, including those of Roberts, Bestor, Wilson, Trautner, Jeltsch etc., clearly defined the "layout" of C5MTases in which a set of relatively short motifs (I-X) combined with a Target Recognition Domain (TRD) typically in between motifs VIII and IX. In the case of the multi-specific MTases from Trautner's lab, the TRD region was shown to be expanded to accommodate several DNA recognition sites (an example would be the M.SPR enzyme which recognises, EcoRII, HaeIII and MspI sites). There are also unorthodox enzymes where the sequence is fragmented reconfigured as heterodimers: look at M.AquI and M.EcoHK31I and one example in which a TRD can be accommodated at separate locations within the polypeptide chain (see M.(phi)BssHII again from Thomas Trautner's lab). Often, these oddball enzymes can help us understand the constraints on structure function relationships amongst enzymes. 

Drawing on many genome data sets, sequence alignments via BLAST, have upheld the original insights from the Roberts and Trautner labs: the catalytic domain contains motifs I-VII, with a connection to motif X, providing a bridge to the TRD (small subdomain), which interacts with motif IX (indicated by NS, left)  to promote the flipping of the correct cytosine into the "jaws" of the catalytic Cys residue (motif IV), that is poised for attack, in part thanks to the stereochemical help from the adjacent Pro residue. As you will all know, the Pro-Cys dipeptide motif is a hallmark of these enzymes.

What do we still want to know? There are a number of groups (Norbert Reich's lab is one example), who are using state of the art biophysical methods to explore the kinetic mechanism and the structural elements in DNA MTases that drive the base flipping reaction. In addition, structural groups, such as John Tainer's lab at Scripps, are developing insight into generic aspects of base flipping (among other things!) from structural and associated biochemical data. You should all be aware of the developments in this area and keep an eye on the literature. However, my own approach has been informed by the many mutants that we and others have studied (in our case with M.MspI, M.SPRI and M.HhaI in particular). In short, we know that the chemistry of methyl transfer (from SAM to the C5 of cytosine, see right) follows from the coordinated flipping of the target base into the active site and I don't think we have anything to add to this. However, this isn't an end to the story.

Perhaps because of my (slavish?) devotion to structure-function relationships in Biochemistry, it came as no surprise to me that Laila's application of the mutagenic Pho polymerase developed by Qaiser and then Sam (from an original piece of work in Bernard Connolly's lab), demonstrated that the role of the conserved motifs in the catalytic mechanism is robust (well at least the active site Cys is essential and loss of other conserved amino acids reduces activity, but rarely abolishes it). However, probably more interestingly (to me), when Laila applied the mutagenesis strategy to the TRD region, she revealed the essential nature of a large number (of unexpected) residues. This of course is now one of our major interests and I will develop this in a broader context of enzyme action in a further Blog.

Back Online

The site has been passive for some time and I thought it would be timely to reactivate it. The main purpose of the blog site is to provide lab members with my own personal views on their data and how it relates to my awareness of the literature, past and present. All information is open access and it is intended to stimulate discussion and exchange of ideas. I will begin with discussions on our methylation work currently in progress.

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

Nature. 2006 Mar 22;
Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, Ignatchenko A, Li J, Pu S, Datta N, Tikuisis AP, Punna T, Peregrín-Alvarez JM, Shales M, Zhang X, Davey M, Robinson MD, Paccanaro A, Bray JE, Sheung A, Beattie B, Richards DP, Canadien V, Lalev A, Mena F, Wong P, Starostine A, Canete MM, Vlasblom J, Wu S, Orsi C, Collins SR, Chandran S, Haw R, Rilstone JJ, Gandi K, Thompson NJ, Musso G, St Onge P, Ghanny S, Lam MH, Butland G, Altaf-Ul AM, Kanaya S, Shilatifard A, O'shea E, Weissman JS, Ingles CJ, Hughes TR, Parkinson J, Gerstein M, Wodak SJ, Emili A, Greenblatt JF

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Who would be a scientist?

"The average trajectory for a successful scientist is the following:

1. age 18-22: paying high tuition fees at an undergraduate college
2. age 22-30: graduate school, possibly with a bit of work, living on a stipend of $1800 per month
3. age 30-35: working as a post-doc for $30,000 to $35,000 per year
4. age 36-43: professor at a good, but not great, university for $65,000 per year
5. age 44: with young children at home (if lucky), fired by the university ("denied tenure" is the more polite term for the folks that universities discard), begins searching for a job in a market where employers primarily wish to hire folks in their early 30s"

Read the rest here.

If you think it is fiction, buy me a pint of Landlord and I'll explain. (Sorry the advert is not PC, but then it is from Keighley).

Histone demethylation by a family of JmjC domain-containing proteins.

Nature. 2006 Feb 16; 439(7078): 811-6
Tsukada Y, Fang J, Erdjument-Bromage H, Warren ME, Borchers CH, Tempst P, Zhang Y

Covalent modification of histones has an important role in regulating chromatin dynamics and transcription. Whereas most covalent histone modifications are reversible, until recently it was unknown whether methyl groups could be actively removed from histones. Using a biochemical assay coupled with chromatography, we have purified a novel JmjC domain-containing protein, JHDM1 (JmjC domain-containing histone demethylase 1), that specifically demethylates histone H3 at lysine 36 (H3-K36). In the presence of Fe(ii) and alpha-ketoglutarate, JHDM1 demethylates H3-methyl-K36 and generates formaldehyde and succinate. Overexpression of JHDM1 reduced the level of dimethyl-H3-K36 (H3K36me2) in vivo. The demethylase activity of the JmjC domain-containing proteins is conserved, as a JHDM1 homologue in Saccharomyces cerevisiae also has H3-K36 demethylase activity. Thus, we identify the JmjC domain as a novel demethylase signature motif and uncover a protein demethylation mechanism that is conserved from yeast to human.

Canadian Outlier

Cold, light snow in Ottawa. Free wireless from the hotel lobby.
We are to brainstorm what will be the next big / useful ideas in the post-genomic era. Maynard Olsen in coming to set the scene.
On complexes, someone in this noble group should look up a few Kd for strong interactions. I never cease to wonder about why everyone homes in on the interactions we might be missing by TAP, rather than enjoying the feast on the table. The British way I suppose.

SAP25

Identification and Characterization of SAP25, a Novel Component of the mSin3 Corepressor Complex.

Mol Cell Biol. 2006 Feb; 26(4): 1386-97
Shiio Y, Rose DW, Aur R, Donohoe S, Aebersold R, Eisenman RN

The transcriptional corepressor mSin3 is associated with histone deacetylases (HDACs) and is utilized by many DNA-binding transcriptional repressors. We have cloned and characterized a novel mSin3A-binding protein, SAP25. SAP25 binds to the PAH1 domain of mSin3A, associates with the mSin3A-HDAC complex in vivo, and represses transcription when tethered to DNA. SAP25 is required for mSin3A-mediated, but not N-CoR-mediated, repression. SAP25 is a nucleocytoplasmic shuttling protein, actively exported from the nucleus by a CRM1-dependent mechanism. A fraction of SAP25 is located in promyelocytic leukemia protein (PML) nuclear bodies, and PML induces a striking nuclear accumulation of SAP25. An isotope-coded affinity tag quantitative proteomic analysis of the SAP25 complex revealed that SAP25 is associated with several components of the mSin3 complex, nuclear export machinery, and regulators of transcription and cell cycle. These results suggest that SAP25 is a novel core component of the mSin3 corepressor complex whose subcellular location is regulated by PML.

IMACS review

Identification of phosphorylation sites using microimmobilized metal affinity chromatography.

Methods Enzymol. 2005; 405: 66-81
Corthals GL, Aebersold R, Goodlett DR

One of the most important roles that mass spectrometry (MS) has played in the late twentieth and early twenty-first centuries has been to assist in the growth of knowledge of dynamic phosphorylation events. Not only has MS allowed researches to pinpoint the site of phosphorylation, but it has also enabled them to identify the kinase/phosphatase pairs responsible for regulation of a specific modification as well as to follow the functional consequences of the observed phosphorylation events on the biology of the system. For phosphorylation analysis, the important contribution of MS has been critical but not definitive. There are numerous methods that have been applied with success, yet none are generally applicable to all analyses. So, for the time being, researchers in the field must select from a panel of methods to find (de)phosphorylation events. In the work described in this chapter, a collection of integrated methods are presented. A detailed account is provided for phosphorylation capture via on- and off-line immobilized metal affinity chromatography (IMAC). This is followed by a suite of useful strategies for discovery of phosphorylation positioning through sequence determination by phosphate-specific diagnostic ion scans, including precursor and product ion scans, neutral loss scans, and in-source dissociation and post-source decay.

The Blog as a Community

My GMail spam has jumped sharply in the past week, with some getting through, so I am wary about entering a new "space" such as this. This post serves to check out some parameters. I use NewsCrawler to set up feeds from my favorite authors, some themes, and some journals. Most journals now offer RSS and/or Atom feeds. I will send a separate post showing its use. Comment spamming is as rife as e-mail spamming, so I suggest you set comments to either none, or to the named authors. A human, but not machine readable text, is used as a password for each comment. Maybe you have done this. I would also turn off posting hornbylab blog to google's feed. Only Dave has these editor features. One limitation may turn out to be the absence of categories, but if it looks like lab blogging is useful, then a move to a hosted WordPress may be appropriate. This would also allow pre-publication or sensitive material to be posted. WordPress is more fully featured and is also free. I can show you next time I am in.
There was an article in the FT about lecturers using podcasts and webcasts of their lectures, a microbiologist at Warwick getting rave reviews! Wireless link webcast and you would not even have to turn up, lecturer or student. The F-floor lecture theatre perhaps marks the end of an era.
To Ottawa tomorrow, from +30 deg to -10 deg in 48 hours. Not good.

BTW, I think you have the clock set to US time.

protein complexes 1

By way of an introduction, the aim of the site is to create a log of experimental work. The information will be made public through Google, so we should make sure that anything pre-publication that is sensitive is not revealed too soon. On the other hand, since most of the experimental work is not of this kind and the sharing of ideas and methods etc is more likely to stimulate our research, I would encourage free exchange between us. If in doubt email me first.

To begin with, there is a need to consider how we develop our thinking and experimental approaches to defining protein complexes. Mainly how do we address the issue of interactors over contaminants? And how do we design functional experiments to test the model of any of our complexes. How do we deal with stable complexes and those that are much more dynamic. Any thoughts? Any key references? Any comments on the recent Nature paper from Cellzome?