Occupancy of sites of phosphorylation in inactive rat heart pyruvate dehydrogenase phosphate in vivo.

1. Inactive pyruvate dehydrogenase phosphate complexes were partially purified from hearts of fed, starved or alloxan-diabetic rats by using conditions that prevent phosphorylation or dephosphorylation. 2. Unoccupied sites of phosphorylation were assayed by incorporation of 32P from [gamma-32P]ATP into the complexes. Total sites of phosphorylation were assayed by the same method after complete reactivation, and thus dephosphorylation, of complexes by incubation with pyruvate dehydrogenase phosphate phosphatase. Occupancy is assumed from the difference (total sites--unoccupied sites). Percentage incorporation into individual sites was measured by high-voltage electrophoresis after tryptic digestion. 3. Values (means +/- S.E.M., in nmol of phosphate/unit of inactive complex) for total sites, occupied sites and percentage occupancies, with numbers of observations in parentheses were: fed, 2.1 +/- 0.04, 1.15 +/- 0.04, 54.8 +/- 1.6% (39); starved, 2.05 +/- 0.03, 1.85 +/- 0.03, 90.2 +/- 1.4% (28); alloxan-diabetic, 1.99 +/- 0.03, 1.72 +/- 0.03, 86.4 +/- 1.4% (68%). 4. Values (means +/- S.E.M. for percentage occupancy) for individual sites of phosphorylation in pyruvate dehydrogenase phosphate given in the order sites 1, 2 and 3 were : fed, 100 +/- 2.7, 27.8 +/- 1.6, 33.9 +/- .9; starved, 100 +/- 1.4, 76.2 +/- 2.0, 92.4 +/- 1.5; alloxan-diabetic, 100 +/- 1.2, 64.0 +/- 1.7, 94.6 +/- 1.4. 5. It is concluded that starvation or alloxan-diabetes leads to a 2--3-fold increase in the occupancy of phosphorylation sites 2 and 3 in pyruvate dehydrogenase phosphate in rat heart in vivo.     

Prediction of Protein Binding to DNA in the Presence of Water-Mediated Hydrogen Bonds

We extend our previous analysis of binding specificity of DNA-protein complexes to complexes containing water-mediated bridges. Inclusion of water bridges between phosphate and base, phosphate and sugar, as well as proteins and DNA, improves the prediction of specificity; six data sets studied in this paper yield correct predictions for all base pairs that have two or more hydrogen-bonds. Beside massive computation, our approach relies highly on experimental data. After deriving protein structures from DNA-protein complexes in which coordinates were established by X-ray diffraction techniques, we analysed all possible DNA sequences to which these proteins might bind, ranking them in terms of Lennard-Jones potential for the optimal docking configuration. Our prediction algorithm rests on the following assumptions: (1) specificity comes mainly from direct hydrogen bonding; (2) electrostatic forces stabilise DNA-protein complexes and contribute only weakly to specificity since they occur at the charged phosphate groups; (3) Van der Waals forces and electrostatic interactions between positively charged groups on the protein and phosphates on DNA can be neglected as they contribute primarily to the free energy of stabilisation as opposed to specificity.     

Fe(III).ATP complexes. Models for ferritin and other polynuclear iron complexes with phosphate

Polynuclear iron complexes of Fe(III) and phosphate occur in seawater and soils and in cells where the iron core of ferritin, the iron storage protein, contains up to 4500 Fe atoms in a complex with an average composition of (FeO.OH)8FeO.OPO3H2. Although phosphate influences the size of the ferritin core and thus the availability of stored iron, little is known about the nature of the Fe(III)-phosphate interaction. In the present study, Fe-phosphate interactions were analyzed in stable complexes of Fe(III).ATP which, in the polynuclear iron form, had phosphate at interior sites. Such Fe(III).ATP complexes are important not only as models but also because they may play a role in intracellular iron transport and in iron toxicity; the complexes were studied by extended x-ray absorption fine structure, EPR, NMR spectroscopy, and measurement of proton release. Mononuclear iron complexes exhibiting a g' = 4.3 EPR signal were formed at Fe:ATP ratios less than or equal to 1:3, and polynuclear iron complexes (Fe greater than or equal to 250, EPR silent at g' = 4.3) were formed at an Fe:ATP ratio of 4:1. No NMR signals due to ATP were observed when Fe was in excess (Fe:ATP = 4:1). Extended x-ray absorption fine structure analysis of the polynuclear Fe(III).ATP complex was able to distinguish an Fe-P distance at 3.27 A in addition to the octahedral O at 1.95 A and 4-5 Fe atoms at 3.36 A. The Fe-O and Fe-Fe distances are the same as in ferritin, and the Fe-P distance is analogous to that in another metal-ATP complex. An observable Fe-P environment in such a large polynuclear iron cluster as the Fe(III).ATP (4:1) complex indicates that the phosphate is distributed throughout rather than merely on the surface, in contrast to earlier models of chelate-stabilized iron clusters. Complexes of Fe(III) and ATP similar to those described here may form in vivo either as normal components of intracellular iron metabolism or during iron excess where the consequent alteration of free nucleotide triphosphate pools could contribute to the observed toxicity of iron.     

Phosphoserine and specific types of its coordination in copper(II) and adenosine nucleotides systems - Potentiometric and spectroscopic studies

Ternary systems of copper(II) complexes with phosphoserine and adenosine 5′-monophosphate or adenosine 5′-diphosphate or adenosine 5′-triphosphate have been investigated. The studies have been performed in aqueous solution using the potentiometric method with computer analysis of the data, ¹³C and ³¹P nuclear magnetic resonance, visible and electron paramagnetic resonance spectroscopies. The overall stability constants of the complexes have been determined. Analysis of the equilibrium constants of the reaction have allowed determination of the effectiveness of the phosphate groups and donor atoms of heterocyclic rings in the process of complex formation. The potential reaction centres are the nitrogen atoms N(1) and N(7) and the phosphate group of the nucleotides as well as phosphate, carboxyl and amine groups from phosphorylated serine. Coordination sites of investigated ligands have been identified on the basis of the equilibrium constants analysis and spectroscopic studies.     

Characterization of vanadate-based transition-state-analogue complexes of phosphoglucomutase by spectral and NMR techniques

Near ultraviolet spectral studies were conducted on two inhibitor complexes obtained by treating the dephospho form of the phosphoglucomutase.Mg2+ complex with inorganic vanadate in the presence of either glucose 1-phosphate [cf. Percival, M. D., Doherty, K., & Gresser, M. J. (1990) Biochemistry (first of four papers in this issue)] or glucose 6-phosphate. Part of the spectral differences between the two inhibitor complexes arises because the glucose phosphate moiety in the complex derived from glucose 1-phosphate binds to the enzyme in a different way from the glucose phosphate moiety in the complex derived from glucose 6-phosphate and because these alternative binding modes produce different environmental effects on the aromatic chromophores of the dephospho enzyme. These spectral differences are strikingly similar to those induced by the binding of glucose 1-phosphate and glucose 6-phosphate to the phospho enzyme--which shows that the glucose 1-phosphate and glucose 6-phosphate moieties occupy positions in the inhibitor complexes closely related to those that they occupy in their respective catalytically competent complexes. This binding congruity indicates that in the inhibitor complexes the oxyvanadium grouping is bound at the site where (PO3-) transfer normally occurs. 31P NMR studies of the phosphate group in these complexes also provide support for this binding pattern. A number of other systems based on compounds with altered structures, such as the deoxysugar phosphates, or systems with different compositions, as in the case of the metal-free enzyme or of the glucose phosphates plus nitrate, also were examined for evidence that complexes analogous to the inhibitor complexes were formed, but none was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of phosphate uptake in corn roots by aluminum-fluoride complexes

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlF(x)) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlF(x) reflects their activity as Pi analogs. For this purpose, (32)P-labeled phosphate uptake by excised roots and plasma membrane H(+)-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlF(x). In vitro, AlF(x) competitively inhibited the rate of root phosphate uptake as well as the H(+)-ATPase activity. Conversely, pretreatment of seedlings with AlF(x) in vivo promoted no effect on the H(+)-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlF(x) pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent (32)Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root (32)Pi uptake induced by AlF(x) pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF(3) and AlF(4)(-) among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.     

Reverse and forward reactions of carbamyl phosphokinase from Streptococcus faecalis R. Participation of nucleotides and reaction mechanisms

The participation of Mg complex of nucleoside diphosphates and nucleoside triphosphates in the reverse and forward reactions catalyzed by purified carbamyl phosphokinase (ATP : carbamate phosphotransferase, EC 2.7.2.2) of Streptococcus faecalis R, ATCC-8043 were studied. The results of initial velocity studies of approx. 1 mM free Mg2+ concentration have indicated that in the reverse reaction MgdADP was as effective a substrate as MgADP. The phosphoryl group transfer from carbamyl phosphate to MgGDP, MgCDP and MgUDP was also observed at relatively higher concentrations of the enzyme and respective magnesium nucleoside diphosphate. In the forward direction MgdATP was found to be as efficient a phosphate donor as MgATP. On the other hand, Mg complexes of GTP, CTP and UTP were ineffective even at higher concentrations of the enzyme and respective magnesium nucleoside triphosphate. Product inhibition studies carried out at non-inhibitory level of approx. 1 mM free Mg2+ concentration have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate or nucleoside triphosphate and the other for carbamyl phosphate or carbamate, and its reaction with the substrates is of the random type. Further tests of numerical values for kinetic constants have indicated that they are partially consistent with the Haldane relationship which is characteristic of rapid equilibrium and random mechanism.     

Primary and predicted secondary structures of the caseins in relation to their biological functions

In free solution, the caseins behave as non-compact and largely flexible molecules with a high proportion of residues accessible to solvent. Historically, they have been described as random coil-type proteins with only a nutritional function. Nevertheless, secondary structure prediction algorithms indicate that many parts of the (unphosphorylated, unglycosylated) polypeptide chains can form regular structures. In particular, a recurrent motif of the Ca2+-sensitive caseins in man, rat, mouse, guinea pig and ruminant species is an alpha-helix--loop--alpha-helix conformation in which the loop region typically contains a cluster of sites of phosphorylation. The biological function of the caseins is considered and it is suggested that the potential or actual conformations of the group of Ca2+-sensitive caseins are suited to the function of modulating the precipitation of calcium phosphate from solution. Either they can act as sites for nucleation or they can bind rapidly to calcium phosphate nuclei as they form spontaneously from supersaturated solution.     

Structure of bovine milk calcium phosphate determined by x-ray absorption spectroscopy

Calcium in cow's milk is mainly in the form of calcium phosphate-phosphoprotein complexes known as casein micelles. These micelles, in contrast to other phosphoprotein complexes in bone and other tissues, can be readily isolated and studied, but conventional techniques have given ambiguous and conflicting evidence on the structure of milk calcium phosphate. Extended X-ray absorption fine structure and near-edge structure measurements at the newly commissioned Synchrotron Radiation Source at Daresbury indicate that it closely resembles brushite, CaHPO₄·2H₂O. This result, and chemical analysis, requires that phosphate groups from the matrix phosphoproteins be incorporated in the brushite lattice, probably in the surface, suggesting that these organic phosphate groups act as heterogeneous nucleation sites for phase separation of the calcium phosphate from solution.     

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase. 1. Effects of sulfate and pH. Mg(II) affinity at the two ATP sites

31P NMR measurements were made (at 121.5 MHz and 5 degrees C) on enzyme-bound substrate complexes of 3-phosphoglycerate kinase in order to address three questions pertaining to (i) the integrity of the enzyme-substrate complexes with Mg(II) in the presence of sulfate concentrations typical of those used for crystallization in X-ray studies, (ii) the relative affinities of Mg(II) to ATP bound at the two sites on the enzyme, and (iii) the pH behavior of the different phosphate groups in the enzyme complexes. 31P chemical shift and spin-spin coupling constant changes showed that at concentrations of 0.5 M and higher, sulfate ion interferes with Mg(II) chelation to ATP and ADP free in solution as well as in their enzyme-bound complexes. The effect on enzyme complexes is stronger for the E.MgATP complex than for the E.MgADP complex. Sulfate ion (50 mM) also causes a approximately 0.5 ppm upfield chemical shift of the 31P resonance of enzyme-bound 3-P-glycerate even in the absence of ATP or Mg(II). A quantitative estimate of the dispartate affinities of Mg(II) to ATP bound at the two sites on the enzyme was made on the basis of computer simulation of changes in the line shape of beta-P (ATP) resonance and of changes in 31P chemical shift of the corresponding gamma-P (ATP) in the E.ATP complex with increasing [Mg(II)]. The concentrations of the relevant species that contribute to these 31P NMR signals were computed by assuming independent binding at the two sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methyl acetyl phosphate as a covalent probe for anion-binding sites in human and bovine hemoglobins

An allosteric modulator of oxygen release in human erythrocytes is 2,3-diphosphoglycerate, but bovine erythrocytes apparently utilize chloride for this purpose since they contain little, if any, 2,3-diphosphoglycerate. In order to identify the sites to which these anions bind, the site-specific acetylating agent, methyl acetyl phosphate, has been employed to compete with these allosteric modulators and to mimic their effects on hemoglobin function. With human hemoglobin A, methyl acetyl phosphate competes with 2,3-diphosphoglycerate and acetylates only Val-1(beta), Lys-82(beta), and Lys-144(beta) within or near the cleft that binds this organic phosphate (Ueno, H., Pospischil, M. A., Manning, J. M., and Kluger, R. (1986) Arch Biochem. Biophys. 244, 795). With bovine hemoglobin, the acetylation is competitive with chloride ion. The sites of acetylation in oxy bovine hemoglobin are Met-1(beta) and Lys-81(beta) and for deoxy bovine hemoglobin, they are Val-1(alpha) and Lys-81(beta). Thus, these sites are expected to be involved in the binding of chloride to bovine hemoglobin. Treatment of either human or bovine hemoglobins with methyl acetyl phosphate under anaerobic conditions leads to a lowering of their oxygen affinity and hence the covalent modifier has the same effect on hemoglobin function as the non-covalent regulators, 2,3-diphosphoglycerate and chloride. The Hill's coefficient of hemoglobin is unaffected by treatment with methyl acetyl phosphate. Under aerobic conditions, specifically acetylated bovine hemoglobin also has a lowered oxygen affinity, and human hemoglobin A shows a slight change in its oxygen affinity. In general, bovine hemoglobin is more responsive than human hemoglobin to both chloride and methyl acetyl phosphate; the latter agent results in a permanent covalent labeling of the protein. Therefore, the results support the idea that methyl acetyl phosphate may be a useful probe for deciphering the sites of binding of anions to proteins.     

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.     

An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

Background  

The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step.     

Chromatin remodelling in mammalian cells by ISWI-type complexes--where, when and why?

The specific location of nucleosomes on DNA has important inhibitory or activating roles in the regulation of DNA-dependent processes as it affects the DNA accessibility. Nucleosome positions depend on the ATP-coupled activity of chromatin-remodelling complexes that translocate nucleosomes or evict them from the DNA. The mammalian cell harbors numerous different remodelling complexes that possess distinct activities. These can translate a variety of signals into certain patterns of nucleosome positions with specific functions. Although chromatin remodellers have been extensively studied in vitro, much less is known about how they operate in their cellular environment. Here, we review the cellular activities of the mammalian imitation switch proteins and discuss mechanisms by which they are targeted to sites where their activity is needed.     

The LIM protein Ajuba is recruited to cadherin-dependent cell junctions through an association with alpha-catenin

Cell-cell adhesive events affect cell growth and fate decisions and provide spatial clues for cell polarity within tissues. The complete molecular determinants required for adhesive junction formation and their function are not completely understood. LIM domain-containing proteins have been shown to be present at cell-cell contact sites and are known to shuttle into the nucleus where they can affect cell fate and growth; however, their precise localization at cell-cell contacts, how they localize to these sites, and what their functions are at these sites is unknown. Here we show that, in primary keratinocytes, the LIM domain protein Ajuba is recruited to cadherin-dependent cell-cell adhesive complexes in a regulated manner. At cadherin adhesive complexes Ajuba interacts with alpha-catenin, and alpha-catenin is required for efficient recruitment of Ajuba to cell junctions. Ajuba also interacts directly with F-actin. Keratinocytes from Ajuba null mice exhibit abnormal cell-cell junction formation and/or stability and function. These data reveal Ajuba as a new component at cadherin-mediated cell-cell junctions and suggest that Ajuba may contribute to the bridging of the cadherin adhesive complexes to the actin cytoskeleton and as such contribute to the formation or strengthening of cadherin-mediated cell-cell adhesion.     

Cardiolipin is the membrane receptor for mitochondrial creatine phosphokinase

Treatment of rat heart mitochondria with phosphate or mersalyl releases a number of proteins, including the mitochondrial creatine kinase (mt-CK). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the released proteins showed that phosphate is more selective than mersalyl in releasing mt-CK. The rebinding of mt-CK to mitochondria was selectively inhibited by adriamycin, which complexes membrane-bound cardiolipin. mt-CK activity and binding experiments have shown that intact mitochondria are able to bind approximately twice the amount of mt-CK they originally contain. Liver mitochondria bound heart mitochondria mt-CK to the same extent as creatine kinase-depleted heart mitochondria. mt-CK was bound by liposomes but only if they contained cardiolipin. The binding of mt-CK to cardiolipin-containing liposomes was inhibited by adriamycin. Phosphatidylcholine liposomes reconstituted with the purified ADP/ATP translocator failed to bind mt-CK.     

Divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.     

Crystal structure of monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8

Monofunctional histidinol phosphate phosphatase from Thermus thermophilus HB8, which catalyzes the dephosphorylation of l-histidinol phosphate, belongs to the PHP family, together with the PHP domain of bacterial DNA polymerase III and family X DNA polymerase. We have determined the structures of the complex with a sulfate ion, the complex with a phosphate ion, and the unliganded form at 1.6, 2.1, and 1.8 A resolution, respectively. The enzyme exists as a tetramer, and the subunit consists of a distorted (betaalpha)7 barrel with one linker and one C-terminal tail. Three metal sites located on the C-terminal side of the barrel are occupied by Fe1, Fe2, and Zn ions, respectively, forming a trinuclear metal center liganded by seven histidines, one aspartate, one glutamate, and one hydroxide with two Fe ions bridged by the hydroxide. In the complexes, the sulfate or phosphate ion is coordinated to three metal ions, resulting in octahedral, trigonal bipyramidal, and tetrahedral geometries around the Fe1, Fe2, and Zn ions, respectively. The ligand residues are derived from the four motifs that characterize the PHP family and from two motifs conserved in histidinol phosphate phosphatases. The (betaalpha)7 barrel and the metal cluster are closely related in nature and architecture to the (betaalpha)8 barrel and the mononuclear or dinuclear metal center in the amidohydrolase superfamily, respectively. The coordination behavior of the phosphate ion toward the metal center supports the mechanism in which the bridging hydroxide makes a direct attack on the substrate phosphate tridentately bound to the two Fe ions and Zn ion to hydrolyze the phosphoester bond.     

Protein-protein interactions as a target for drugs in proteomics

Protein-protein interactions play a central role in numerous processes in the cell and are one of the main fields of functional proteomics. This review highlights the methods of bioinformatics and functional proteomics of protein-protein interaction investigation. The structures and properties of contact surfaces, forces involved in protein-protein interactions, kinetic and thermodynamic parameters of these reactions were considered. The properties of protein contact surfaces depend on their functions. The contact surfaces of permanent complexes resemble domain contacts or the protein core and it is reasonable to consider such complex formation as a continuation of protein folding. Characteristics of contact surfaces of temporary protein complexes share some similarities with active sites of enzymes. The contact surfaces of the temporary protein complexes have unique structure and properties and they are more conservative in comparison with active site of enzymes. So they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations were undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or, on the contrary, to induce protein dimerization.     

Structural motif of phosphate-binding site common to various protein superfamilies: all-against-all structural comparison of protein-mononucleotide complexes

In order to search for a common structural motif in the phosphate-binding sites of protein-mononucleotide complexes, we investigated the structural variety of phosphate-binding schemes by an all-against-all comparison of 491 binding sites found in the Protein Data Bank. We found four frequently occurring structural motifs composed of protein atoms interacting with phosphate groups, each of which appears in different protein superfamilies with different folds. The most frequently occurring motif, which we call the structural P-loop, is shared by 13 superfamilies and is characterized by a four-residue fragment, GXXX, interacting with a phosphate group through the backbone atoms. Various sequence motifs, including Walker's A motif or the P-loop, turn out to be a structural P-loop found in a few specific superfamilies. The other three motifs are found in pairs of superfamilies: protein kinase and glutathione synthetase ATPase domain like, actin-like ATPase domain and nucleotidyltransferase, and FMN-linked oxidoreductase and PRTase.