Analysis of water patterns in protein kinase binding sites

Deregulation of protein kinases is associated with numerous diseases, making them important targets for drug discovery. The majority of drugs target the catalytic site of these proteins, but due to the high level of similarity within the ATP binding sites of protein kinases, it is often difficult to achieve the required pharmacological selectivity. In this study, we describe the identification and subsequent analysis of water patterns in the ATP binding sites of 171 protein kinase structures, comprising 19 different kinases from various branches of the kinome, and demonstrate that structurally similar binding sites often have significantly different water patterns. We show that the observed variations in water patterns of different, but structurally similar kinases can be exploited in the structure-based design of potent and selective kinase inhibitors.     

Structure and functional analysis of the fungal galectin CGL2

Recognition of and discrimination between potential glyco-substrates is central to the function of galectins. Here we dissect the fundamental parameters responsible for such selectivity by the fungal representative, CGL2. The 2.1 A crystal structure of CGL2 and five substrate complexes reveal that this prototype galectin achieves increased substrate specificity by accommodating substituted oligosaccharides of the mammalian blood group A/B type in an extended binding cleft. Kinetic studies on wild-type and mutant CGL2 proteins demonstrate that the tetrameric organization is essential for functionality. The geometric constraints due to the orthogonal orientation of the four binding sites have important consequences on substrate binding and selectivity.     

Identification of residues involved in water versus glycerol selectivity in aquaporins by differential residue pair co-evolution

Aquaporins (AQPs) are members of the Major Intrinsic Protein (MIP) family that can transport water or glycerol, as well as other compounds. The rationale for substrate selectivity at the structural level is still incompletely understood. The information present in multiple sequence alignments (MSAs) can help identify both structural and functional features, especially the complex networks of interactions responsible for water or glycerol selectivity. Herein, we have used the method of Statistical Coupling Analysis (SCA) to identify co-evolving pairs of residues in two separate groups of sequences predicted to correspond to water or glycerol transporters. Differentially co-evolved pairs between the two groups were tested by their efficacy in correctly classifying a training set of MSAs, and binary classifiers were built with these pairs. Up to 50% of the residues found in hundreds of binary classifiers corresponded to only ten positions in the MSA of aquaporins. Most of these residues are close to the lining of the aquaporin pore and have been identified previously as important for selectivity. Therefore, this method can shed light on the residues that are important for substrate selectivity of aquaporins and other proteins. SCA requires a very large sequence dataset with relatively low homology amongst its members, and these requirements are met by aquaporins.     

Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures.

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4, 1518-1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34-3.5 and 3.3-0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.     

Substrate binding is a prerequisite for stabilisation of mouse thymidine kinase in proliferating fibroblasts

Thymidine kinase (TK) expression in mammalian cells is strictly growth regulated, with high levels of the enzyme present in proliferating cells and low levels in resting cells. We have shown that mouse TK expressed from a constitutive promoter is still subject to this regulation. The drastic decline in TK enzyme levels in resting cells is largely due to a pronounced reduction in the half-life of the protein. Deletion of the 30 C-terminal amino acid residues from TK abrogates growth regulation, rendering the enzyme very stable. Moreover, the substrate thymidine was sufficient to stabilise the labile TK protein in quiescent cells. Here, we report that the ability of TK to bind substrates is essential for both growth-dependent regulation and stabilisation by the substrate. By mutation or elimination of the binding sites for either of the two substrates, ATP and thymidine, we expressed TK proteins lacking enzymatic activity which abolished growth-regulated expression in both cases. Mutant TK proteins impaired in substrate binding were subject to rapid degradation in exponentially growing cells and thymidine was no longer sufficient to inhibit this rapid decay. A C-terminal truncation known to stabilise the TK wild-type protein in resting cells did not affect the rapid turnover of enzymatically inactive TK proteins. Proteasome inhibitors also failed to stabilise these substrate-binding mutants. By cross-linking experiments, we show that TK proteins with mutated substrate-binding sites exist only as monomers, whereas active TK enzyme forms dimers and tetramers. Our data indicate that, In addition to the C terminus intact substrate-binding sites are required for growth-dependent regulation of TK protein stability.     

Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements

Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.     

Binding sites of ubiquitin-protein ligase. Binding of ubiquitin-protein conjugates and of ubiquitin-carrier protein

It was found previously that the enzyme ubiquitin-protein ligase (E3) contains specific protein substrate binding sites that are responsible for the selection of proteins for degradation by the ubiquitin system. In the present study, we have tried to gain more insight into the mode of action of E3 by the characterization of other binding sites of this enzyme. Following the ligation of ubiquitin to 125I-lysozyme, the conjugates produced are very tightly bound to E3, as indicated by size analysis on glycerol density gradient centrifugation. The strong binding of ubiquitin-protein conjugates to the enzyme may account for the apparently processive addition of multiple molecules of ubiquitin to the protein substrate. Both the protein substrate moiety and the ubiquitin moiety participate in the interaction of ubiquitin-protein conjugates with E3, as indicated by competition with specific agents and by the comparison of the binding of ubiquitin-conjugated protein to that of free protein. In addition to the binding of its substrates and products, E3 also appears to interact with some of the enzymes with which it acts in concert. When E3 is incubated with the ubiquitin-carrier protein E2, a complex is formed between the two enzymes as analyzed on glycerol gradients. The formation of an E2.E3 complex may facilitate the transfer of activated ubiquitin from E2 to the protein substrate bound to the ligase.     

Recognition of double-stranded RNA by proteins and small molecules

Molecular recognition of double-stranded RNA (dsRNA) is a key event for numerous biological pathways including the trafficking, editing, and maturation of cellular RNA, the interferon antiviral response, and RNA interference. Over the past several years, our laboratory has studied proteins and small molecules that bind dsRNA with the goal of understanding and controlling the binding selectivity. In this review, we discuss members of the dsRBM class of proteins that bind dsRNA. The dsRBM is an approximately 70 amino acid sequence motif found in a variety of dsRNA-binding proteins. Recent results have led to a new appreciation of the ability of these proteins to bind selectivity to certain sites on dsRNA. This property is discussed in light of the RNA selectivity observed in the function of two proteins that contain dsRBMs, the RNA-dependent protein kinase (PKR) and an adenosine deaminase that acts on dsRNA (ADAR2). In addition, we introduce peptide-acridine conjugates (PACs), small molecules designed to control dsRBM-RNA interactions. These intercalating molecules bear variable peptide appendages at opposite edges of an acridine heterocycle. This design imparts the potential to exploit differences in groove characteristics and/or base-pair dynamics at binding sites to achieve selective binding.     

Squash glycerol-3-phosphate (1)-acyltransferase. Alteration of substrate selectivity and identification of arginine and lysine residues important in catalytic activity

Glycerol-3-phosphate 1-acyltransferase is a soluble chloroplast enzyme involved in glycerol-lipid biosynthesis associated with chilling resistance in plants (). Resistance is associated with higher selectivity for unsaturated acyl substrates over saturated ones. In vitro substrate selectivity assays performed under physiologically relevant conditions have been established that discriminate between selective and non-selective forms of the enzyme. A mutation, L261F, in the squash protein converts it from a non-selective enzyme into a selective one. The mutation lies within 10 A of the predicted acyl binding site and results in a higher K(m) for 16:0 acyl carrier protein (ACP). Site-directed mutagenesis was used to determine the importance of four residues, Arg(235), Arg(237), Lys(193), and His(194), implicated to be involved in binding of the phosphate group of glycerol 3-phosphate to the enzyme. All the proteins were highly homologous in structure to the wild type enzyme. Mutations in Arg(235), Arg(237), and Lys(193) resulted in inactive enzyme, while His(194) had reduced catalytic activity. The mutant proteins retained the ability to bind stoichiometric quantities of acyl-ACPs supporting the potential role of these residues in glycerol 3-phosphate binding.     

Evidence that cyclic nucleotides activating rabbit muscle protein kinase I interact with both types of cAMP binding sites associated with the enzyme

Eighty different adenine-modified cAMP analogs were tested as activators of rabbit muscle protein kinase I (cAKI) in an in vitro phosphotransferase assay. All the analogs tested were able to activate completely the kinase. The affinities of the cAMP derivatives for the two types (A and B) of binding sites associated with the regulatory moiety of cAKI were determined under conditions similar to those used in the phosphotransferase assay. The potency of the cAMP analogs as cAKI activators was found to correlate with the mean affinity for sites A and B, rather than to the affinity for only one of the sites. This was true whether cAKI was assayed at low or near physiological ionic strength, whether the concentration of cAKI binding sites was 0.2 or 400 nM, and whether the kinase substrate was mixed histones or homogeneous phenylalanine-4-monooxygenase. Furthermore, site A-selective and site B-selective cAMP analogs activated cAKI synergistically. Finally, it was shown that the degree of synergism between cAMP analogs in activating cAKI correlated with their degree of site selectivity. It is concluded that cyclic nucleotides interact with both types of binding sites in the process of cAKI activation.     

Switch from an aquaporin to a glycerol channel by two amino acids substitution

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.     

Architecture and selectivity in aquaporins: 2.5 a X-ray structure of aquaporin Z

Aquaporins are a family of water and small molecule channels found in organisms ranging from bacteria to animals. One of these channels, the E. coli protein aquaporin Z (AqpZ), has been shown to selectively conduct only water at high rates. We have expressed, purified, crystallized, and solved the X-ray structure of AqpZ. The 2.5 A resolution structure of AqpZ suggests aquaporin selectivity results both from a steric mechanism due to pore size and from specific amino acid substitutions that regulate the preference for a hydrophobic or hydrophilic substrate. This structure provides direct evidence on the molecular mechanisms of specificity between water and glycerol in this family of channels from a single species. It is to our knowledge the first atomic resolution structure of a recombinant aquaporin and so provides a platform for combined genetic, mutational, functional, and structural determinations of the mechanisms of aquaporins and, more generally, the assembly of multimeric membrane proteins.     

The Structural Basis of ATP as an Allosteric Modulator

Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems.     

Phosphorylation of high molecular weight proteins in platelets treated with 12-O-tetradecanoylphorbol-13-acetate

Changes in the phosphorylation of three high molecular weight cytoskeletal proteins in platelets (actin binding protein, platelet talin and myosin heavy chain) were investigated after treatment with a phorbol ester. All three showed rapid increases in phosphate incorporation, reaching near-maximal values within three minutes. Phosphopeptide maps of the proteins before and after phorbol treatment revealed a single new site in myosin heavy chain, two new peptides in actin binding protein, and multiple sites in talin. These results point to multiple cytoskeletal targets of protein kinase C and suggest complex mechanisms for reorganizing microfilaments.     

Hepatic microsomal glucuronidation of bilirubin in unilamellar liposomal membranes. Implications for intracellular transport of lipophilic substrates

It has been assumed that following hepatic uptake, bilirubin is bound exclusively to cytosolic proteins prior to conjugation by microsomal UDP-glucuronyl-transferase. Since bilirubin partitions into lipid rather than the aqueous phase at neutral pH, we postulated that bilirubin reaches the sites of glucuronidation by rapid diffusion within membranes. To examine this hypothesis, [14C]bilirubin was incorporated into the membrane bilayer of small unilamellar liposomes of egg phosphatidylcholine. Radiochemical assay of this membrane-bound substrate in a physiologic concentration, using native rat liver microsomes, demonstrated immediate formation of bilirubin glucuronides at a more rapid initial velocity than for bilirubin bound to the high-affinity sites of purified cytosolic binding proteins, i.e. glutathione S-transferases (p less than 0.025) or native liver cytosol (p less than 0.05). Kinetic analysis suggested that the mechanisms of substrate transfer from liposomal membranes and from purified glutathione S-transferases to microsomal UDP-glucuronyltransferase were similar. The exchange of 3H- and 14C-labeled bilirubin substrate between binding proteins and liposomal membranes was then investigated using Sepharose 4B chromatography. As the concentration of bilirubin was increased relative to that of protein, net transfer of substrate from the protein to the membrane pool was observed. These findings indicate that bilirubin is efficiently transported by membrane-membrane transfer to hepatic microsomes, where it undergoes rapid conjugation. Bilirubin entering hepatocytes may partition between membrane and cytosolic protein pools, but as intracellular bilirubin concentration increases, the membrane pool is likely to provide a greater proportion of the substrate for glucuronidation.     

Exploring sequence-structure relationships in the tyrosine kinome space: functional classification of the binding specificity mechanisms for cancer therapeutics

MOTIVATION: Evolutionary and structural conservation patterns shared by more than 500 of identified protein kinases have led to complex sequence-structure relationships of cross-reactivity for kinase inhibitors. Understanding the molecular basis of binding specificity for protein kinases family, which is the central problem in discovery of cancer therapeutics, remains challenging as the inhibitor selectivity is not readily interpreted from chemical proteomics studies, neither it is easily discernable directly from sequence or structure information. We present an integrated view of sequence-structure-binding relationships in the tyrosine kinome space in which evolutionary analysis of the kinases binding sites is combined with computational proteomics profiling of the inhibitor-protein interactions. This approach provides a functional classification of the binding specificity mechanisms for cancer agents targeting protein tyrosine kinases. RESULTS: The proposed functional classification of the kinase binding specificities explores mechanisms in which structural plasticity of the tyrosine kinases and sequence variation of the binding-site residues are linked with conformational preferences of the inhibitors in achieving effective drug binding. The molecular basis of binding specificity for tyrosine kinases may be largely driven by conformational adaptability of the inhibitors to an ensemble of structurally different conformational states of the enzyme, rather than being determined by their phylogenetic proximity in the kinome space or differences in the interactions with the variable binding-site residues. This approach provides a fruitful functional linkage between structural bioinformatics analysis and disease by unraveling the molecular basis of kinase selectivity for the prominent kinase drugs (Imatinib, Dasatinib and Erlotinib) which is consistent with structural and proteomics experiments.     

The carbohydrate-binding site in galectin-3 is preorganized to recognize a sugarlike framework of oxygens: ultra-high-resolution structures and water dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 ? at 100 K, 1.25 ? at 298 K) and in complex with lactose (0.86 ?) or glycerol (0.9 ?). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.     

Unique MAP Kinase binding sites

Map kinases are drug targets for autoimmune disease, cancer, and apoptosis-related diseases. Drug discovery efforts have developed MAP kinase inhibitors directed toward the ATP binding site and neighboring "DFG-out" site, both of which are targets for inhibitors of other protein kinases. On the other hand, MAP kinases have unique substrate and small molecule binding sites that could serve as inhibition sites. The substrate and processing enzyme D-motif binding site is present in all MAP kinases, and has many features of a good small molecule binding site. Further, the MAP kinase p38alpha has a binding site near its C-terminus discovered in crystallographic studies. Finally, the MAP kinases ERK2 and p38alpha have a second substrate binding site, the FXFP binding site that is exposed in active ERK2 and the D-motif peptide induced conformation of MAP kinases. Crystallographic evidence of these latter two binding sites is presented.     

pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model

Background  

Protein kinase A (cAMP-dependent kinase, PKA) is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal.     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.