Tuesday, November 23, 2010

Update on structures

First an update on the last one:
The structure of EF-Tu on the ribosome has now been solved with a non-hydrolyzable guanine nucleotide, in the true activated state. The metal ion that I guessed might be there is in fact not, but the ordered region of the effector loop is, in fact, ordered. This will hopefully lay to rest the idea that the elongation factors are activated by DISordering of their active site loops, which was always the biggest eyesore of all the models for their function so far. And the contacts with the ribosome are surprisingly simple, involving mainly just two histidines acting as a "ruler" to measure the orientation of the rRNA phosphate backbone. One is aligned in a phosphate-imidazole-water network vaguely reminiscent of the serine protease catalytic triad, explaining enhanced catalysis.

In addition, the number of G-protein coupled receptor structures has been steadily increasing. A friend asked me when I thought that solving new inactive GPCR structures would become boring, and while I'm sure this point will come sooner than anyone would predict at this time, we still have a ways to go. The family of seven-transmembrane receptors is very diverse, and there will no doubt be peculiarities to some of the more atypical members, like metabotropic glutamate receptors or even weirder ones like Frizzled. Yes, I know the last is not technically a GPCR, though it can be made into one quite easily by merely mutating loops, so I think of it as one. And that's not to mention the 100+ orphan receptors, whose ligand identification may be accelerated if we had structures.
Read more!

Tuesday, June 15, 2010

A possible mechanism for elongation factors--finally?

Like the last speculation, this one involves the ribosome. In particular, the two elongation factors that in bacteria are known as EF-Tu and EF-G. Like all GTPases, these proteins have a catalytic domain that resembles the Ras family of signaling molecules. Clearly, however, the details of the catalysis are distinct. In common with each other, both types of proteins have low catalytic activity on their own (though the quantitative meaning of "low" varies"), and need to be activated by binding to something else in order to achieve rapid catalysis.

In the case of Ras-like proteins, these partners are called GTPase-activating proteins, or GAPs, and they bind to a particular region of the proteins near the phosphates of the bound GTP. Their activity commonly (though not always) involves an arginine side chain that is inserted into the active site and presumably stabilizes the transition state. However, EF-Tu and EF-G are activated by interacting with a particular state of the ribosome. The sarcin-ricin loop, or SRL, of the large subunit occupies a position similar to GAPs in Ras-like GTPases, but in the available structures it makes few contacts to the protein. This is probably due to the fact that one part of the protein, the so-called Switch I segment, is disordered.

Some researchers have suggested that the disordering of this segment is what triggers catalysis, but I am very skeptical of that, largely for the simple reason that I've never seen another enzyme where catalysis is facilitated by decreasing the number or closeness of contacts to the substrate. In fact, the common scenario is the opposite, where loop or domain closures activate enzymes by making the contacts more extensive. I rather suspect that the disorder observed is due to the inability to actually form and trap the GTP-bound, activated form in the crystal. But in any case, the "arginine finger" mechanism, or similar, cannot operate, as there are no positively charged groups of any kind protruding from the SRL.

An intriguing (relatively) recent paper suggested that a GTPase involved in 30S ribosomal subunit assembly, YqeH, uses a potassium ion coordinated near the GTP phosphates to replace the arginine used by Ras. This was based on a previous structural demonstration that a related protein involved in tRNA modification uses this mechanism. Interestingly, both of these GTPases interact with folded RNA in one way or another, which raises the question of whether the elongation factors could similarly use a second metal ion (in addition to the Mg2+ common to all GTPases) to trigger catalysis upon interaction with the SRL. In fact, there is an acidic Asp residue in the P-loop region of both EFs near where this hypothetical cation would have to be.

However, a quick inspection of the structures of EF-Tu and EF-G bound to the ribosome shows that even at the modest resolution, a direct participation of the SRL in the coordination sphere of such an ion is essentially ruled out, at least in the approximate relative positioning of the molecules observed. Therefore, any role of the SRL in electrostatically recruiting a cation would need to be water-mediated. Alternatively, the Switch I region of GTP-bound EF-Tu contains a short helix whose dipole points very roughly toward the Asp in the P-loop, and realignment of this helix upon ribosome binding may provide a way to recruit a cation only upon proper juxtaposition of the two. As no structure exists of EF-G with the switch segments ordered, it is unknown whether this helix exists in that protein as well. It seems that even if a second cation is involved in the mechanism of elongation factors, it will be very difficult to obtain a structure with it present.
Read more!

Tuesday, May 18, 2010

Kids and race

A non-biochemistry post for a change--I'm sure most of you know about the study that shows how much racial bias children demonstrate when asked to judge pictures of people with different skin color. I wanted to respond to the following comment made by author Po Bronson:

"(Parents) want to give their kids this sort of post-racial future when they're very young and they're under the wrong conclusion that their kids are colorblind. ... It's in the absence of messages of tolerance that they will naturally ... develop these skin preferences."

I have to question the use of the word "naturally" here. It seems it is being used to really mean "even though their parents don't teach them to"--when in reality there are plenty of other sources they could pick up this prejudice from, especially other kids. Even young children appear not to be immune to the tendency to take cues from peers in terms of who is "good" and "bad", and be more likely to like a popular than an unpopular classmate.

The study does nothing to determine where these kids picked up their light skin preference, but it surely isn't some sort of simple effect of perceptual familiarity, as the black children showed it too. To call it "natural" is to make it look as though racial tolerance is this difficult thing to master, and that parents are solely to blame when it is lacking.
Read more!

Thursday, March 4, 2010

Fold.it

Today I found an interesting site called Foldit, where you can download a program that lets you try folding a protein graphically. It differs from sites like Folding@home, which just draw on your computer's spare processor cycles, in that it actually hopes to use human intuition to help solve the protein folding problem, and compare it to the performance of computer algorithms on the same task. I downloaded it, and wanted to give my opinion on it.

First for the pros:
I am very happy (and pleasantly surprised) by the overall idea, that of finding humans who have unique mental capabilities that may be useful for solving real-world problems in biology. It often seems that the only kind of unusually gifted minds (whether you call them prodigies, savants, etc.) that most people know about, or at least care about, are "Rainman"-like human calculators, and then only as some kind of "freak show". I do believe there well could be people who have an intuitive sense of the forces stabilizing proteins, and could fold at least small proteins intuitively, just science never hears of them. And it's not just with this problem by any means. I think there are lots of optimization sort of problems in all areas of science and technology where certain humans could outperform computers--yet the dogma in the field is that there is no use in humans trying to do these tasks themselves.

In the modern world of the internet, I think the time has very much come for people who know or suspect they have such unique talents to network with each other, and share both ideas and life experiences. Whether it is to actually figure out how to use those skills in the real world, or just to relate to the feeling of alienation that arises when one feels his/her thinking style is not accepted by others, this is a definite niche for a social networking site. In fact, it was exactly this type of group I was looking for when I stumbled across Foldit. In case you're wondering, I did not find any such groups, so I will probably be starting one in the not too distant future

But back to Foldit, I quickly realized there are many things that make it less than ideal, both as a way to discover hidden talent and as a means of solving the folding problem:
1) Lack of transparency:
When one looks at the ranking system for users, it becomes clear that the judgment criterion is not similarity to some known native structure, but score by a Rosetta-type scoring function. The specific function used, however, is obscure. If this were just some video game, I could see the makers wanting to keep the exact scoring system secret, in order to not make the game too easy. However, if this is even pretending to be science, then a very open disclosure of the nature of the problem is in order, even if the scoring function is very realistic. One might worry that using a real native structure as the reference would allow cheating by those who know the structure--but CASP has found a logical way around this, by using unpublished structures.
2) Manipulation is unwieldy:
The only way to fold a protein is to bend the backbone by dragging it from one point, and then the rest follows in a "springy" fashion. There's no good way to "tweak" individual residues. Also, there should be some sort of way to specify helix formation and beta-sheet pairing on some global level, and then build these elements as "ideal" units that can then be bent to allow packing.
3) Side chain handling is weird:
Side chains do not have full mobility, they "spring" only to common rotamers that are reasonably clash-free. While this can be useful, especially for beginners, as it prevents really bad things like sticking a methionine through the "hole" in a benzene ring, it also means that it is not possible to stretch a side chain to form, e.g., a hydrogen bond, and then allow the structure to relax with this already made. That goes for everything, in fact--while interactive molecular mechanics is way cool, it's very good to be able to turn OFF the force field, to allow getting out of local minima. Especially when the mechanics is not "real" molecular mechanics. Also, Asp/Asn and Glu/Gln are impossible to tell apart, and the "flip" of Asn/Gln amides is invisible. This is not great for folding, and downright awful for ligand modeling (see below).
4) Ligands are included, but almost definitely not well:
A few of the tutorial levels involve moving a non-protein ligand into position. This was probably introduced to allow enzyme/receptor design. However, one must wonder what kind of force field is used to describe protein-ligand interactions. The whole idea of Rosetta is very "globalist", and for something like ligand recognition, which could be dominated by precise hydrogen bonding and stacking geometries, it is unlikely that this program does it justice. Add to that the insistence that every side chain be in a common rotamer at all times, and it is not even possible to move them into ligand-centric positions. So, the utility of this feature is questionable, in my opinion.

So the conclusion is, two thumbs up for the effort to give human intuition a chance to hack away at real scientific problems. But, this program may well do a rather poor job at this, as well as in predicting structures. It was rather easy to get within the top five scores for some proteins, and yet I did not feel as if I was actually creating good-looking structures. Most likely, the top scorers will be those who can best get around the awkwardness of being stuck in local minima by the limited controls. And if you want to test your ability to predict structures for your own sake (as opposed to impressing others), maybe downloading old CASP targets is a better bet.

Read more!

Thursday, January 14, 2010

Just a crazy idea (warning, technical)

This is officially the first time I actually say something "serious" about science on this blog. I don't know if there are any who will read this who will have the knowledge to even understand this, but I mainly want to record this somewhere in the off case that it turns out to be correct.

Anyway, I have had a long-standing interest in the ribosome, and recently I read a structural paper in Cell reporting the structure of the ribosome in complex with a ribonuclease called RelE. This is a heck of a weird RNase, as it cuts messenger RNA as it is being translated, in the middle of a codon that's about to be read.

The authors show very convincingly that the RNA is NOT cleaved between the first and second nucleotides of the codon, but that it is cleaved between the second and third nucleotide. In the structure, this site lines up with a site on RelE that corresponds to the active site in a number of related RNases, and that's all well and good.

On the other hand, there's a small (or maybe big) problem here. All possible catalytic residues are more than 5 Angstroms from the scissile bond in both ribosomes, except maybe for Arg 81, which is mutated to Ala. In fact, there is not a single contact made to this phosphate. Furthermore, the 2' -OH nucleophile is positioned in a stereochemically awkward manner, that would require a large rotation of the phosphate to allow attack opposite the 5' -OH leaving group (See Figure 4B in the paper). In contrast, in the related nuclease (Fig. 4A), if you take the phosphate oxygen near the catalytic His 92 as the leaving group mimic, the 2' -OH of the guanine is a nice 180 degrees from it. While a rotation is certainly not impossible, and may be hindered here by the 2' O-methyl modification of the adenine, it seems it would require a significant movement of the guanine ribose.

What is most intriguing, though, is that in one ribosome the next phosphate, the one separating the current A-site codon from the next, appears better poised for attack. Not only would the required rotation of the phosphate be only about 60 degrees, rather than 120 degrees for the preceding phosphate, but this could be well accommodated without much movement of either ribose. In addition, if the 2' -OH were not methylated, it would probably move to hydrogen bond with the N3 of C 1054. Finally, the residue Arg 56 is both near the 2' -OH and the leaving group.

This immediately suggests that there may have been a primordial ribozyme-like activity in which C 1054 activated the 2' -OH of the third nucleotide for attack on its phosphate, and was assisted by some group playing the role of Arg 56, both helping activate the nucleophile and promote leaving group departure. In fact, when I had viewed the pre-cleavage state structure before reading the biochemical evidence for the scissile bond being in the other position, and not being familiar with other RNases, I just assumed that this was the scissile phosphate in the modern RelA.

In turn, this prompted the (maybe crazy) idea that possibly such an activity remains even today, and when the mRNA is cleaved after the third base, this makes the third base (i.e., the G) more mobile, allowing it to better align to be cut again at the site identified by the authors. While such a complicated cascade "violates" Occam's razor, its advantage would be that it provides a way to guard against excessive activity of a potentially toxic enzyme like RelA, particularly if the first cut were reversible.

Unfortunately, only the 5' fragment was isolated. If two successive cuts were made, this fragment would be the same, but the 3' fragment would be one nucleotide shorter, and a free 2'-3' cyclic GMP would be formed. This may be totally off, but on the other hand the thought was too striking not to write down somewhere.
Read more!

Wednesday, April 29, 2009

The Swine Flu Song

I haven't posted on here in a long time, but I just saw this and got an idea that I wanted to share with the world.

A San Francisco man named Stephan Zielinski, who is NOT a biochemist, decided to turn the sequence of swine flu hemagglutinin into a song.

Someone totally needs to take his algorithm and incorporate it into a plugin for one of the structure viewers like Chimera. Then, artists can create audiovisual works in which not only is a protein "played" musically, but the side chain of each amino acid flashes in the 3-D structure simultaneously with its respective sound. In this way, a linear piece of music is made to correspond with a path through 3-D space... How mindblowing.
Read more!

Wednesday, July 9, 2008

Membrane protein structures--part 3

I haven't posted here for a long time, so I decided to summarize a little of membrane structural biology over the last few months.

By far the most exciting advancements have occurred in the field of GPCRs. The Stevens group at The Scripps Research Institute (who also published the adrenoceptor structure mentioned a few posts ago) published a new structure of the same receptor, but with a different antagonist (3D4S). In this structure, a cholesterol molecule was observed in a slightly different location from in the previous one, which led to the discovery of a putative cholesterol recognition motif. Also, Schertler and colleagues at the MRC laboratory in Cambridge solved the structure of the turkey beta1 adrenoceptor with yet a third antagonist (2VT4).



One might think that with the first structures of "real" pharmacologically relevant GPCRs being solved, the relevance of rhodopsin, the previously prototypical member of the superfamily, might be on the way out. However, this is most definitely not a conclusion to jump to automatically, as a recent structure (3CAP) from Park et. al. demonstrates. This is the structure of opsin, which is rhodopsin without the covalently attached light-absorbing molecule retinal. Many of the helices show a change in conformation relative to rhodopsin, and based on some biochemical data showing constitutive activity in opsin, the authors propose that this structure may represent the long-sought active state of the receptor. This structure shows changes of a significantly greater magnitude from dark state rhodopsin than any other structure previously proposed to be activated, and this makes me more inclined to believe that it could actually be active. In fact, I am guessing the shift observed in opsin may be TOO large to represent normal activation, and may represent a partial unfolding of the receptor due to complete loss of the stabilizing effect of retinal. Though much less notable, for the sake of completeness I will add that a structure of squid rhodopsin has also been published (2Z73).

As for ion channels, a bacterial homologue of the Cys-loop superfamily, to which nicotinic and GABA(A) receptors in animals belong, has been solved (2VL0). The ligand of this receptor is unknown, and the low homology with other Cys-loop receptors along with the mediocre resolution mean that this structure will most likely not be useful for modeling studies. Also, this is not recent, but the structure of the acid-sensing channel ASIC1 (2QTS) provides insight into a new fold of ion channel. The most intriguing property of this structure is the fact that the homotrimeric channels show each monomer to be in a different conformation, with some of the differences being large (almost 10 Angstroms). This may be an artifact of the crystal structure, but if not, this would be an unprecedented finding.

In the area of transporters, not that much has happened, although there is a structure of a sodium-galactose transporter by a group at UCLA due to be released soon. Intriguingly, this transporter is related to LeuT, despite a lack of sequence similarity aside from conservation of a few glycines at proposed hinge regions. The fact that this transporter is in the inward-facing conformation, as opposed to LeuT, which was in the outward-facing conformation, should provide insight into the transport mechanism.

Read more!