From Protein Structure to Function with Bioinformatics.pdf

262 R.A. Laskowskiaccessibility and electrostatic potential (Shanahan et al. 2004). Any positive hits,therefore, may indicate that the target protein is a DNA binder, although, of course,this says little more about the protein’s function than that.Our example structure, 2fck, returned no HTH hits – as one would expect.10.3.1.3 NestsThe third method searches for any nest motifs in the structure. These are frequentlyassociated with functional sites. A nest is an anion or cation binding siteformed by three or more amino acids in the sequence whose main chain ψ-ϕ dihedralangles alternate between the right- and left-handed helical (α and γ) regionsof the Ramachandran plot (Watson and Milner-White 2002a, b). Again, a RasMolview shows the location of the nest in the context of the whole 3D structure.ProFunc assigns a score to each nest based on: the number of NH atoms that areaccessible to solvent, the conservation scores of its constituent residues, andwhether the nest occurs in one of the larger clefts on the surface. Nests can beuseful when none of the other methods have much to say about the protein’s function.In these cases, nests can suggest locations on the 3D structure which may befunctionally important.The 2fck structure contains several nests, three of which score highly enough toindicate that they may be functionally significant. And indeed, the top-scoring nestis in the protein’s likely substrate binding site (based on the similarity identifiedabove to the 1s7f structure), while nests two and three are found at the entrance tothe coA binding site.10.3.1.4 Surface CleftsNext, all the clefts in the protein’s surface are computed, using the SURFNETprogram (Laskowski 1995). The clefts are ranked in order of size and can beviewed in RasMol. The viewing options allow for the cleft surfaces to be colouredby specific properties, such as cleft size, residue type or residue conservationscore. Cleft size is important as the largest cleft in a protein’s surface tends to bewhere the protein’s active site is located (Laskowski et al. 1996). Also importantis residue conservation, as clusters of highly conserved residues, particularly iflocated in a large pocket, are highly indicative of a functional site (Lichtarge andSowa 2002; Madabushi et al. 2002; Glaser et al. 2003). Like nest analysis, studyof the protein’s surface clefts is of most use when the other methods have failed orhave suggested only vague possibilities. And it can also suggest functionallyimportant parts of the structure.In our example structure, the largest cleft does indeed correspond to the protein’sputative binding site, matching the location of the bound coA in the related structuresidentified from the fold-match above and the template methods described next.