Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes.

A detailed comparison of the structures of aspartate aminotransferase, alanine race-mase, the beta subunit of tryptophan synthase, D-amino acid aminotransferase and glycogen phosphorylase has revealed more extensive structural similarities among pyridoxal phosphate (PLP)-binding domains in these enzymes than was observed previously. These similarities consist of seven common structural segments of the polypeptide chain, which form an extensive common structural organization of the backbone chain responsible for the appropriate disposition of key residues, some from the aligned fragments and some from variable loops joined to these fragments, interacting with PLPs in these enzymes. This common structural organization contains an analogous hydrophobic minicore formed from four amino acid side chains present in the two most conserved structural elements. In addition, equivalent alpha-beta-alpha-beta supersecondary structures are formed by these seven fragments in three of the five structures: alanine racemase, tryptophan synthase and glycogen phosphorylase. Despite these similarities, it is generally accepted that these proteins do not share a common heritage, but have arisen on five separate occasions. The common and contiguous alpha-beta-alpha-beta structure accounts for only 28 residues and all five enzymes differ greatly in both the orientation of the PLP pyridoxal rings and their contacts with residues close to the common structural elements.

Finding local structural similarities among families of unrelated protein structures: a generic non-linear alignment algorithm.

We have developed a generic tool for the automatic identification of regions of local structural similarity in unrelated proteins having different folds, as well as for defining more global similarities that result from homologous protein structures. The computer program GENFIT has evolved from the genetic algorithm-based three-dimensional protein structure comparison program GA_FIT. GENFIT, however, can locate and superimpose regions of local structural homology regardless of their position in a pair of structures, the fold topology, or the chain direction. Furthermore, it is possible to restrict the search to a volume centered about a region of interest (e.g., catalytic site, ligand-binding site) in two protein structures. We present a number of examples to illustrate the function of the program, which is a parallel processing implementation designed for distribution to multiple machines over a local network or to run on a single multiprocessor computer.

Two "unrelated" families of ATP-dependent enzymes share extensive structural similarities about their cofactor binding sites.

Two proteins, D-alanine:D-alanine ligase and cAMP-dependent protein kinase, share a remarkable degree of structural convergence despite having different three-dimensional folds and different enzymatic functions. Here we report that as many as 103 residues from 10 segments form two identical super-secondary structures between which the cofactor ATP is bound. The cofactor, two bound metal cations, and several water molecules form a large network of electrostatic and hydrophobic interactions common to both enzymes, and these are mediated by the similar placement of equivalent amino acids within the common supersecondary structures.

Enzyme-mononucleotide interactions: three different folds share common structural elements for ATP recognition.

Three ATP-dependent enzymes with different folds, cAMP-dependent protein kinase, D-Ala:D-Ala ligase and the alpha-subunit of the alpha2beta2 ribonucleotide reductase, have a similar organization of their ATP-binding sites. The most meaningful similarity was found over 23 structurally equivalent residues in each protein and includes three strands each from their beta-sheets, in addition to a connecting loop. The equivalent secondary structure elements in each of these enzymes donate four amino acids forming key hydrogen bonds responsible for the common orientation of the "AMP" moieties of their ATP-ligands. One lysine residue conserved throughout the three families binds the alpha-phosphate in each protein. The common fragments of structure also position some, but not all, of the equivalent residues involved in hydrophobic contacts with the adenine ring. These examples of convergent evolution reinforce the view that different proteins can fold in different ways to produce similar structures locally, and nature can take advantage of these features when structure and function demand it, as shown here for the common mode of ATP-binding by three unrelated proteins.