COMPUTER MODELING OF AN ATP-BINDING PROTEIN

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The origins of water-soluble proteins appear to be considerably more difficult to identify than the origins of membrane-bound proteins. Most transmembrane proteins, even those that are functionally and structurally complex, are built of a small number of structural elements that are shared across protein families. Moreover, there are simple, natural or synthetic models consisting of the same elements that can perform essential membrane-related functions. This is not the case with cytoplasmic proteins. In contemporary cells, they are usually quite large by protobiological standards, but in contrast to membrane proteins they cannot be significantly reduced in size without loss of activity. Using a novel in vitro technique, Keefe and Szostak selected ATP-binding proteins from six trillion random polypeptides. They found four new protein families, each containing proteins with highly similar amino acid sequences that were unrelated to each other or to anything found in the current protein databases. The frequency of finding ATP-binding proteins appears to be similar to the frequency of finding ATP-binding ribozymes. Proteins from one family have been characterized in fair detail. The originally selected protein contained 80 amino acids but deletion studies revealed that the minimal binding unit is less than 50 amino acids long and, thus, is the smallest known ATP-binding protein. The proteins are highly selective towards ATP and its close analog, adenosine diphosphate (ADP), as they bind neither guanosine triphosphate (GTP) nor cyclic AMP. However, their sequences do not contain any already identified ATP-binding motifs. To function, they require zinc ions and contain four conserved cysteine residues. More recently, the high resolution, three-dimensional structure of a protein from the family was solved using X-ray crystallography. As all biological, water-soluble proteins, this structure has a hydrophobic core, but exhibits a novel fold. It consists of a three-stranded antiparallel beta-sheet and two nonadjacent alpha-helices. ADP is stabilized in the binding pocket by stacking interactions with phenylalanine and tyrosine residues and by hydrogen bonds to several side chains in the protein. Selectivity of binding appears to be insured by hydrogen bonds between the N1, N3 and N6 of adenine and methianine-45 and glycine-63. A zinc ion is coordinated by the conserved cysteines in a region not adjacent to the binding pocket. The ATP-binding protein is a very interesting protobiological model because it is the first example of a simple, functional protein that has not been a subject of long evolutionary optimization. However, its folding pattern may be evolutionarily deficient. For example, it may not have the capability to acquire new specificity through mutations. We propose to examine the protein from this point of view and, if necessary, redesign its sequence in an attempt to eliminate the deficiencies without altering the fold. If this task were successful it would lead to the creation of a novel fold that appears to be suitable for evolution, thus providing an empirical argument supporting an "evolutionary accident" hypothesis of the origin of enzymes. If we found that the sequence could not be appropriately redesigned it would suggest that the fold, even if it were present among protobiological proteins, was not likely to survive subsequent evolutionary pressures. Although it would be clearly premature to draw conclusions from a single negative example, this result would hint that a hypothesis about evolutionary pruning of protein structures is worth serious considerations. In either case, we would gain an understanding how to construct and identify good candidate models for evolutionarily viable protobiological enzymes.