Principles of Protein Structure - H. Schulz 1982

Protein–ligand interactions
Phosphoryl group binding sites

There is no single, universal phosphoryl-binding center in Proteins. Approximately 50% of the studied proteins bind or otherwise interact with compounds bearing phosphoryl groups. Contrasting Examples such as Ribonuclease [689], human Hemoglobin (which attaches 2,3-diphosphoglycerate, ATP, or Inositol hexaphosphate in the position between the two ß-chains [672]), and staphylococcal nuclease [242] demonstrate that proteins do not possess a unique, universal phosphoryl-binding site.

The loop between the first strand of the ß-sheet in the Rossmann fold and the subsequent a-helix serves as a characteristic phosphoryl-binding center. There is a group of proteins in which a phosphoryl or pyrophosphoryl moiety attaches at a characteristic site. This site is a polypeptide chain loop that connects the first* pleated strand of a parallel ß-sheet and a helix located approximately antiparallel to this sheet. Typically, this loop mimics the loop between the first ß-strand of the Rossmann fold and the adjacent a-helix (Fig. 5.12, b).

Phosphoryl groups are anchored relative to the backbone of the loop by Hydrogen Bonds. In S-malate dehydrogenase [691], D-glyceraldehyde-3-phosphate dehydrogenase [230], and Lactate dehydrogenase [230], the pyrophosphate moiety of NAD is hydrogen-bonded to the main chain of the corresponding loop. In the case of Alcohol dehydrogenase [692], binding studies using an NAD analog showed that the pyrophosphate resides in the same position (within 3 Å) relative to this loop. In flavodoxin, the loop is "wrapped" around the phosphoryl moiety of FMN [145, 237, 238]. As substrate binding studies indicate, in adenylate kinase the loop backbone is wrapped around the AMP phosphate, while in crystalline adenylate kinase, this loop fixes a sulfate ion from the mother liquor [665]. ATP binds in an analogous position in phosphoglycerate kinase [310, 311]. In Glutathione reductase [124], the pyrophosphoryl group of NADP attaches to the backbone of the corresponding Rossmann fold loop. The same applies to the FAD pyrophosphate. In Triosephosphate isomerase [305], the phosphate is also located near the loops connecting the ß-sheets and subsequent a-helices, but at the C-terminal region of the polypeptide chain (Fig. 5.17, d). In all these instances, the loop between the carbonyl end of the ß-sheet and the succeeding a-helix is utilized. This can be explained by the favorable electrostatic interaction between the negative charge of the phosphoryl group and the a-helix dipole [792], which arises from the superposition of Hydrogen bond dipoles.

* It should be recalled that ß-sheets are numbered according to their position along the linear polypeptide chain; residues belonging to the first sheet lie closer to the N-terminus.

Phosphoryl-binding loops can be flexible and mobile. Several other generalizations emerge from the examples considered. In all cases, phosphoryl groups form hydrogen bonds with the polypeptide backbone. Phosphoryl binding is carried out by loops projecting from a rigid secondary or supersecondary Structure, such as the Rossmann fold. Because these loops have conformationally rigid bases, they can themselves exhibit mobility without disrupting the overall Cell/13.html">Protein Structure. In porcine adenylate kinase, for instance, the phosphate-binding loop has the sequence Gly-Pro-Gly-Ser-Gly-Lys-Gly [389]; the presence of glycyl residues at every second position allows for a wide range of dihedral angles at the C_a atoms (sec. 2.3). Consequently, this chain segment can adopt a broad spectrum of Conformations, resulting in flexibility and mobility even in the non-interacting enzyme molecule. X-Ray Structural Analysis (Fig. 10.5) has shown that upon the transition of adenylate kinase from conformation A to conformation B, this loop undergoes a 6 Å shift.

The similarities within the aforementioned group of phosphoryl-binding proteins may not necessarily share a common origin. Regarding the dehydrogenases described in sec. 10.4, the mode of (pyro)phosphoryl group binding is likely a conserved feature, as these proteins appear to be homologous (sec. 9.6). However, for other proteins in this group, the phosphoryl binding mode cannot be regarded as an indicator of Homology, as it may simply be a consequence of favorable physicochemical interactions.

Some proteins modified by covalent phosphorylation share typical properties common to nucleotide-binding proteins. There is A large number of proteins whose Functions are modified or regulated via reversible phosphorylation [175]. Amino acid sequences at the phosphorylation sites of various proteins exhibit certain similarities (Table 10.2). Typically, the side chain of a Ser, Thr, or His residue is phosphorylated. Often, a Gly residue is positioned immediately before or right after such a residue; in most cases, the residue two positions further down the chain turns out to be a positively charged Lys or Arg. These sequences bear certain similarities to the phosphoryl-binding loop of adenylate kinase (Table 10.2). The hypothesis that reversible phosphorylation of a given residue governs Conformational Changes in the phosphoprotein [175, 693] correlates well with the fact that in lactate dehydrogenase [232] and adenylate kinase [688], the loop serves as the epicenter of major conformational changes. Thus, studies of nucleotide-binding proteins can help elucidate the static and dynamic aspects of protein phosphorylation.

Class="center">Table 10.2 Phosphate attachment sites in nucleotide-binding proteins and Phosphoproteins