Ssu72 Discussion: Difference between revisions

From MDWiki
Jump to navigationJump to search
Line 15: Line 15:
===Serine Phosphatase?===
===Serine Phosphatase?===


Based on the very high primary ( )and secondary ( ) structural similarities between the human and Drosophila Ssu72s, and, most importantly, the near perfect superimposition of one on the other in DaliLite ( ), we can suppose, for the purposes of this analysis, that the two have identical functional characteristics. On the basis of that proposition, it is worthwhile to discuss the evidence which supports their role as a serine phosphatase.
Based on the very high primary ( )and secondary ( ) structural similarities between the human and Drosophila Ssu72s, and, most importantly, the near perfect superimposition of one on the other in DaliLite ( ), we can suppose, for the purposes of this analysis and without further experimentation, that the two have identical functional characteristics. On the basis of that proposition, it is worthwhile to discuss the evidence which supports their role as a serine phosphatase.


===References===
===References===

Revision as of 13:00, 14 June 2009

Function

In attempting to infer the function of the Drosophila Ssu72 protein, two broad sources of information were used, each leading to distinct conclusions (the compatibility of which will be considered in due course). The value of the first data set as a proxy for experimental information about the functionally uncharacterised Drosophila protein is predicated on the high level of similarity in amino acid sequence and almost identical match in both secondary and 3D structure that were found to exist between this protein and the human Ssu72 protein. This data comprises both in vivo and in vitro functional analyses of the latter protein, which tend to suggest that the protein in Drosophila similarly functions as a serine phosphatase – presumably at the C-terminal domain of RNA polymerase II (Krishnamurthy et al., 2004) – and has a role in 3' end mRNA processing that is independent of its activity as a phosphatase. The second piece of guidance comes from the results generated by the use of computational and bioinformatic tools in this paper, which prima facie reveal the Drosophila protein to be a protein tyrosine phosphatase. Therefore, these two seemingly conflicting conclusions must now be evaluated on the basis of the limited evidence available to determine which is more likely to be true, or whether one necessarily precludes the other.

Protein Tyrosine Phosphatase?

An evaluation of the Drosophila protein as a potential Protein Tyrosine Phosphatase (PTPase) is a logical place to begin this discussion as this is what Meinhart, Silberzahn, & Cramer (2003) concluded about the human protein, based on their initial characterization of its primary, secondary and 3D structures, as well as a number of important pieces of experimental data. They began by identifying, within the amino acid sequence of the Ssu72 gene, the presence of a PTPase signature motif at the N-terminal end of the protein. This motif is a short sequence, the consensus sequence being (H/V)C(X5)R(S/T), which forms the phosphate-binding site (P-loop) in the mature protein and is where, invariably, members of the PTPase superfamily exert their catalytic action. This motif is similarly present in the Drosophila Ssu72 protein and, what is more, shares almost the precise sequence as that of the human protein (VCSSNQNRS), but for an unknown amino acid in place of the glutamine (Q). Meinhart, Silberzahn, & Cramer (2003) inferred, based on this motif, that if the human Ssu72 were a PTPase, it would be one of the low molecular weight family. This inference was based on the presence of a characteristic asparagine (N) in the motif, which the Drosophila protein shares. Further analysis of the amino acid sequence of the human protein found the presence of two important residues, which form part of an 'aspartate loop' that is important for the binding of substrate at the P-loop. Those residues are aspartates 140 and 143, both of which are present in the Drosophila protein and which were found, by mutational analysis, to contribute to the human protein's activity.


In the functional results presented in this report, ProFunc was used to, among other things, identify potential, functionally-important triad template motifs from a database. The only such motif that was found to be present in the Drosophila protein was centered around the three important residues of the PTPase signature motif - valine 8, cysteine 9 and serine 16 - the latter two of which are essential. The 3-dimensional coordinates that those residues occupy in the protein were found to match those of a low molecular PTPase (myobacterium tuberculosis low molecular weight tyrosine phosphatase) with an RSMD of 0.15Å (see Figure 1). The degree to which this motif is conserved is, at least in part, indicative of its physiological importance. This high degree of evolutionary conservation across a range of organisms (see Figure 2), combined with the fact that mutation of the essential cysteine residue to a serine abolishes human Ssu72 activity and is embryonic lethal (Meinhart, Silberzahn, & Cramer, 2003), is extremely strong evidence that this motif is functionally important. According to Zhang (1998), 'amino acid sequence comparisons of the catalytic domains of PTPases with the catalytic subunits of protein Ser/Thr phosphatases have shown no sequence similarity'. It is therefore unlikely that the serine phosphatase activity reported by Krishnamurthy et al. (2004), among others, occurs at this catalytic site; or, if it does, is by a mechanism that has not been characterised thus far.


Meinhardt, Silberzahn, & Cramer (2003) then went on to examine in vitro whether the human protein did indeed function as a tyrosine phosphatase. Their finding that it did was based on it acting in vitro to cleave a phospho-tyrosine analogue, pNPP, and that PTPase inhibitors caused an abolition of this phosphatase activity. It is likely, given the conservation of the PTPase catalytic domain in the Drosophila protein, that this experimental result could be replicated with it. However, it now becomes important to consider how the broad structure of the Drosophila protein compares with that of known PTPases. In the structural results presented in this report, DaliLite was used to superimpose the 3D form of the Drosophila Ssu72 protein over inter alia a representative low molecular weight tyrosine phosphatase - Bovine Low Molecular Weight PTPase (see Figure 3). The match is, on the face of it, quite poor. However, in the absence of more relevant information about which particular regions are shared between the two proteins, it is impossible to determine what the significance of such a difference is. It is possible that it is of no functional consequence as far as potential PTPase activity is concerned, the catalytic motif being present, and that the difference accounts for additional activity lacking in traditional PTPases - such as serine/threonine phosphatase activity. However, more analysis is required to elucidate this more completely.

Serine Phosphatase?

Based on the very high primary ( )and secondary ( ) structural similarities between the human and Drosophila Ssu72s, and, most importantly, the near perfect superimposition of one on the other in DaliLite ( ), we can suppose, for the purposes of this analysis and without further experimentation, that the two have identical functional characteristics. On the basis of that proposition, it is worthwhile to discuss the evidence which supports their role as a serine phosphatase.

References

Zhang Z-Y. 1998 Protein-Tyrosine Phosphatases: Biological Function, Structural Characteristics, and Mechanism of Catalysis. Critical Reviews in Biochemistry and Molecular Biology, 33(1):1–52.

Evolution

Heading

Structure

Heading

Abstract | Introduction | Results | Discussion | Method | References