The beta12-beta13 loop of protein phosphatase-1 is involved in activity regulation

Protein phosphatase-1 (PP1) is a member of the eukaryotic serine/threonine phosphatase gene family. The beta12-beta13 loop is a prominent non-conserved region among the family, and extends from the surface and overhangs the active site. To investigate the function of the beta12-beta13 loop of PP1, we systematically examined all residues by site-directed deletion mutation. Deleting residues Y272, E275 or F276, caused enzyme activity to increase, while deleting residue C273, caused enzyme activity to decrease, when G274 was deleted no remarkable activity increase was observed, and almost all activity was lost when D277, N278 or A279 were deleted. These observations implied that each amino acid has a different effect on the activity of phosphatase, which may result from their different side chains and locations. The activity change of these PP1 mutants, from Y272 to A279, was comparable to that of calcineurin mutants, from Y311 to K318. By comparison, except for D277 (N316) and A279 (K318) of PP1 (calcineurin), each pair of equivalent mutants in the beta12-beta13 loop of PP1 and calcineurin have coincident activity change although they are non-conserved, which suggests that the beta12-beta13 loop of PP1 is not only involved in activity regulation but also involved in regulation similar to that of calcineurin.     

Importance of the beta12-beta13 loop in protein phosphatase-1 catalytic subunit for inhibition by toxins and mammalian protein inhibitors.

Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the beta12-beta13 loop attenuated the inhibition of the resulting PP1alpha catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with dose-response characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1alpha, the PP1alpha catalytic core, and CRHM2 with identical IC(50). Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the beta12-beta13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the beta12-beta13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.     

The beta12-beta13 loop is a key regulatory element for the activity and properties of the catalytic domain of protein phosphatase 1 and 2B

The molecular architectures of the catalytic core of protein phosphatase 1 (PP1) and protein phosphatase 2B (PP2B) are similar, and both contain a beta12-beta13 loop that consists of non-conserved residues. A truncation mutant containing the PP2B catalytic domain has previously been constructed in our laboratory, and designated CNAa. In this study, the PP1 catalytic subunit (PP1c) and CNAa, as well as mutants with the corresponding loops exchanged, were investigated using multiple substrates. Deletion of the beta12-beta13 loop from Y272 to A279 of PP1c or from Y311 to K318 of CNAa resulted in inactive proteins. Loop exchange generated chimeric mutants called PP1-CNAa-loop and CNAa-PP1-loop. The activities and kinetic parameters of the two chimeric mutants were altered in the direction of the enzyme from which its loop was derived. The activity of PP1c or CNAa-PP1-loop was similar whether preincubated with Mn(2+) or not, while CNAa and PP1-CNAa-loop can acquire enhanced activation if preincubated with Mn(2+) for longer periods of time. Intrinsic fluorescence spectra revealed that the three-dimensional structure was altered as a result of exchanging the loops of PP1c and CNAa. In conclusion, the beta12-beta13 loop is one of the key regulatory elements in the catalytic domain for the activity and properties of PP1c and CNAa.     

Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1

Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.     

Tyr306 near the C-terminus of protein phosphatase-1 affects enzyme stability and inhibitor binding

Previous deletion studies have suggested that Tyr306 has an important influence on the properties of protein phosphatase-1 (PP1). To test this inference, we constructed three site-directed mutants, PP1Y306A, PP1Y306K, and PP1Y306E. The specific activity of PP1Y306A was 3.5-fold higher than that of PP1wt, especially with K-R-Tp-I-R-R as substrate, and it also had a 13-fold higher Kcat and a 43-fold higher Kcat /Km . PP1Y306K and PP1Y306E, in that order, and were next in terms of increased activity. Use of the denaturant guanidine hydrochloride (GdnHCl) demonstrated that mutation of this site decreased enzyme stability. PP1Y306A and PP1Y306E lost all activity when incubated for 24 h in 0.6 M GdnHCl, and their fluorescence spectra confirmed the loss of stability. Because all three substitutions had a similar effect, we infer that the aromatic group of Tyr plays a crucial role in maintaining enzyme stability. Our results show that Tyr306 does affect the spatial conformation of the catalytic subunit of PP1 molecule. The IC50 of PP1Y306A for the inhibitor microcystin-LR was threefold higher than that of PP1wt, whereas those of PP1Y306E for tautomycin and norcantharidin were 15-fold and 10-fold higher, respectively. We conclude that Tyr306 plays an important role in enzyme stability and inhibitor binding.     

The predicted beta12-beta13 loop is important for inhibition of PP2Acalpha by the antitumor drug fostriecin

The potential anticancer agent fostriecin (FOS) is a potent inhibitor of the protein Ser/Thr phosphatases PP2A and PP4 and a weaker inhibitor of PP1. Random mutagenesis and automated screening in yeast identified residues in human PP2Acalpha important for inhibitory FOS binding. A C269S substitution in the predicted beta12-beta13 loop decreased the FOS sensitivity of intact cells and increased the IC(50) of PP2Acalpha by 10-fold in vitro. Changing PP2Acalpha Cys-269 to phenylalanine, the equivalent residue in PP1, and the Y267G and G270D substitutions caused a similar effect. The results provide information relevant to the design of novel protein Ser/Thr phosphatase inhibitory drugs.     

Complex role of the beta 2-beta 3 loop in the interaction of U1A with U1 hairpin II RNA

RNA recognition motifs (RRMs) are characterized by highly conserved regions located centrally on a beta-sheet, which forms the RNA binding surface. Variable flanking regions, such as the loop connecting beta-strands 2 and 3, are thought to be important in determining the RNA-binding specificities of individual RRMs. The N-terminal RRM of the spliceosomal U1A protein mediates binding to an RNA hairpin (U1hpII) in the U1 small nuclear RNA. In this complex, the beta(2)-beta(3) loop protrudes through the 10-nucleotide RNA loop. Shortening of the RNA loop strongly perturbs binding, suggesting that an optimal "fit" of the beta(2)-beta(3) loop into the RNA loop is an important factor in complexation. To understand this interaction further, we mutated or deleted loop residues Lys(50) and Met(51), which protrude centrally into the RNA loop but do not make any direct contacts to the bases. Using BIACORE, we analyzed the ability of these U1A mutants to bind to wild type RNAs, or RNAs with shortened loops. Alanine replacement mutations only modestly affected binding to wild type U1hpII. Interestingly, simultaneous replacement of Lys(50) and Met(51) with alanine appeared to alleviate the loss of binding caused by shortening of the RNA loop. Deletion of Lys(50) or Met(51) caused a dramatic loss in stability of the U1A.U1hpII complex. However, deletion of both residues simultaneously was much less deleterious. Simulated annealing molecular dynamics analyses suggest this is due to the ability of this mutant to rearrange flanking amino acids to substitute for the two deleted residues. The double deletion mutant also exhibited substantially reduced negative effects of RNA loop shortening, suggesting the rearranged loop is better able to accommodate a short RNA loop. Our results indicate that one of the roles of the beta(2)-beta(3) loop is to provide a steric fit into the RNA loop, thereby stabilizing the RNA.protein complex.     

Juxtamembrane localization of the protein phosphatase-1 inhibitor protein PHI-1 in smooth muscle cells

Protein phosphorylation regulates many fundamental processes and protein phosphatase-1 (PP1) is a major phosphatase that determines the levels of Ser/Thr phosphorylation. Regulatory subunits and inhibitor phosphoproteins control PP1 activity. PHI-1 is a member of a family of PP1 inhibitor phosphoproteins that was discovered based on sequence similarity to the known inhibitor CPI-17. To learn more about PHI-1 we determined the tissue distribution of PHI-1 in embryonic and adult tissues, and examined its cellular localization by immunohistochemistry. In the embryo PHI-1 appeared first in the heart at E10, and by E15 it was detected in multiple tissues. Expression in adult tissues was strikingly different, with PHI-1 detected primarily in smooth muscles in the intestine, blood vessels, and male and female genitourinary tracts. PHI-1 also was highly expressed in the endothelial layer of blood vessels. Both PHI-1 and CPI-17 are expressed predominantly in adult smooth muscles. Whereas CPI-17 staining was diffuse PHI-1 was concentrated along the cell membrane in distinct foci, detected by confocal and electron microscopy. The common tissue distribution but different cellular localization of PHI-1 and CPI-17 suggest distinctive physiological roles for these two PP1 inhibitors.     

Identification of a novel phosphatidic acid binding domain in protein phosphatase-1

Phosphatidic acid (PA) has been recognized as a lipid second messenger, yet few cellular targets for PA have been identified. Previous work demonstrated PA as a potent and noncompetitive tight-binding inhibitor of the catalytic subunit (gamma isoform) of protein phosphatase-1 (PP1c gamma) in vitro. The high potency of inhibition, coupled with high specificity for PA over other phospholipids, suggested the presence of a high-affinity PA binding domain on PP1c gamma. In the current study, quantification of the binding interaction and identification of the binding domain were pursued. Surface plasmon resonance was employed to quantitate the interaction between PP1c gamma and immobilized mixed lipid vesicles of PA/phosphatidylcholine (PC) or PC alone. The data disclosed a high-affinity interaction with a KD measured in the low (1-40) nanomolar range, consistent with the range of Ki previously obtained from in vitro enzymatic assays. Next, identification of the segment of PP1 necessary for PA binding was determined using a deletion mutagenesis strategy. Binding assays revealed that PP1c gamma residues between 274 and 299 were required for the interaction with the lipid. When fusions of PP1c gamma fragments with green fluorescent protein (GFP) were generated, it was then determined that PP1c gamma residues 286-296 were sufficient to confer PA binding to GFP, a protein that does not interact with PA. The minimal PA binding domain of PP1c gamma lacked similarity to the previously described PA binding segments of Raf-1 kinase and cyclic-AMP phosphodiesterase 4A1. When these results were taken together with the known crystallographic structure of PP1, they identified a novel PA binding region on PP1c gamma that contains a unique loop-strand structural fold responsible for the interaction with PA.     

The role of the C-terminal domain of protein tyrosine phosphatase-1B in phosphatase activity and substrate binding

Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in the regulation of the insulin receptor. Dephosphorylation of the insulin receptor results in decreased insulin signaling and thus decreased glucose uptake. PTP-1B-/- mice have increased insulin sensitivity and are resistant to weight gain when fed a high fat diet, validating PTP-1B as a potential target for the treatment of type 2 diabetes. Many groups throughout the world have been searching for selective inhibitors for PTP-1B, and most of them target inhibitors to PTP-1B-(1-298), the N-terminal catalytic domain of the enzyme. However, the C-terminal domain is quite large and could influence the activity of the enzyme. Using two constructs of PTP-1B and a phosphopeptide as substrate, steady state assays showed that the presence of the C-terminal domain decreased both the Km and the k(cat) 2-fold. Pre-steady state kinetic experiments showed that the presence of the C-terminal domain improved the affinity of the enzyme for a phosphopeptide 2-fold, primarily because the off-rate was slower. This suggests that the C-terminal domain of PTP-1B may contact the phosphopeptide in some manner, allowing it to remain at the active site longer. This could be useful when screening libraries of compounds for inhibitors of PTP-1B. A compound that is able to make contacts with the C-terminal domain of PTP-1B would not only have a modest improvement in affinity but may also provide for specificity over other phosphatases.     

Crystal structure of protein C inhibitor provides insights into hormone binding and heparin activation

Protein C inhibitor (PCI) is a member of the serpin family that has many biological functions. In blood it acts as a procoagulant, and, in the seminal vesicles, it is required for spermatogenesis. The activity of PCI is affected by heparin binding in a manner unique among the heparin binding serpins, and, in addition, PCI binds hydrophobic hormones with apparent specificity for retinoids. Here we present the 2.4 A crystallographic structure of reactive center loop (RCL) cleaved PCI. A striking feature of the structure is a two-turn N-terminal shortening of helix A, which creates a large hydrophobic pocket that docking studies indicate to be the retinoid binding site. On the basis of surface electrostatic properties, a novel mechanism for heparin activation is proposed.     

Stem of SL1 RNA in HIV-1: structure and nucleocapsid protein binding for a 1 x 3 internal loop

The 5'-leader of HIV-1 RNA controls many viral functions. Nucleocapsid (NC) domains of gag-precursor proteins select genomic RNA for packaging by binding several sites in the leader. One is likely to be a stem defect in SL1 that can adopt either a 1 x 3 internal loop, SL1i (including G247, A271, G272, G273) or a 1 x 1 internal loop (G247 x G273) near a two-base bulge (A269-G270). It is likely that these two conformations are both present and exchange readily. A 23mer RNA construct described here models SL1i and cannot slip into the alternate form. It forms a 1:1 complex with NCp7, which interacts most strongly at G247 and G272 (K(d) = 140 nM). This demonstrates that a linear G-X-G sequence is unnecessary for high-affinity binding. The NMR-based structure shows an easily broken G247:A271 base pair. G247 stacks on both of its immediate neighbors and A271 on its 5'-neighbor; G272 and G273 are partially ordered. A bend in the helix axis between the SL1 stems on either side of the internal loop is probable. An important step in maturation of the virus is the transition from an apical loop-loop interaction to a dimer involving intermolecular interactions along the full length of SL1. A bend in the stem may be important in relieving strain and ensuring that the strands do not become entangled during the transition. A stem defect with special affinity for NCp7 may accelerate the rate of the dimer transformation. This complex could become an important target for anti-HIV drug development, where a drug could exert its action near a high-energy intermediate on the pathway for maturation of the dimer.     

Crystal structure of human insulin-like growth factor-1: detergent binding inhibits binding protein interactions

Despite efforts spanning considerably more than a decade, a high-resolution view of the family of proteins known as insulin-like growth factors (IGFs) has remained elusive. IGF-1 consists of three helical segments which are connected by a 12-residue linker known as the C-region. NMR studies of members of this family reveal a dynamic structure with a topology resembling insulin but little structural definition in the C-region. We have crystallized IGF-1 in the presence of the detergent deoxy big CHAPS, and determined its structure at 1.8 A resolution by multiwavelength anomalous diffraction, exploiting the anomalous scattering of a single bromide ion and six of the seven sulfur atoms of IGF-1. The structure reveals a well-defined conformation for much of the C-region, which extends away from the core of IGF-1 and has residues known to be involved in receptor binding prominently displayed in a type II beta-turn. In the crystal, these residues form a dimer interface, but analytical ultracentrifugation experiments demonstrate that at physiological concentrations IGF-1 is monomeric. A single detergent molecule contacts residues known to be important for IGF-1 binding protein (IGFBP) interactions. Biophysical and biochemical data show that the detergent binds to IGF-1 specifically and blocks binding of IGFBP-1 and IGFBP-3.     

The complete primary structure of calcineurin A, a calmodulin binding protein homologous with protein phosphatases 1 and 2A

A complementary DNA (cDNA) clone encoding the catalytic subunit of calcineurin (calcineurin A) has been isolated from a rat brain cDNA library. The primary structure of the cDNA consists of 2,337 nucleotides including the entire coding region for 521 amino acids, and the calculated molecular mass is 58,643 Da. The calcineurin A is strikingly homologous to protein phosphatases 1 and 2A, approximately 50% of the amino acids over an internal 250-residue region between residues 78 and 329 being identical. Twenty four amino acid-residue region between residues 391 and 414 shows the consensus structural features for a calmodulin-binding domain. These data suggest that the allosteric character of this chimeric enzyme is generated by gene fusion of two separate protein families.     

Crystal structure of a conformation-selective casein kinase-1 inhibitor

Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.     

Characterization of the binding of transforming growth factor-beta 1, -beta 2, and -beta 3 to recombinant beta 1-latency-associated peptide.

Preprotransforming growth factor-beta 1 (TGF beta 1) is a 390-amino acid precursor polypeptide that undergoes a number of processing steps to yield mature TGF beta 1 (amino acid residues 279-390) and a pro portion (residues 30-278) termed beta 1-latency-associated peptide (beta 1LAP). The dimeric form of beta 1LAP has been shown to associate noncovalently with the mature growth factor, resulting in inactivation of biological activity. To further characterize this interaction, the mature TGF beta 1 was radioiodinated and used to determine dissociation constants. A cross-linking method using the bifunctional covalent cross-linker bis-(sulfosuccinimidyl)suberate was found to be the best approach for measuring the amount of bound growth factor. The efficiency of cross-linking was constant within each experiment and varied between 45-55%. Saturation plots and their associated Scatchard analyses indicate apparent Kd values between 1.1-1.8 nM. Competition of TGF beta 1 binding to beta 1LAP by TGF beta 2 and TGF beta 3 (two closely related growth factors) revealed that the latter also bind beta 1LAP tightly, with apparent Kd values of 1.9 and 0.4 nM, respectively.     

Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.

The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.     

Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor

Metallo beta-lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of beta-lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo beta-lactamases. Reported here are the crystal structures of the native IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4 -(phenylb utyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 A (native) and 2.0 A (complex) resolution. Binding of the inhibitor in the active site induces a conformational change that results in closing of the flap and transforms the active site groove into a tunnel-shaped cavity enclosing 83% of the solvent accessible surface area of the inhibitor. The inhibitor binds in the active site through interactions with residues that are conserved among metallo beta-lactamases; the inhibitor's carboxylate group interacts with Lys161, and the main chain amide nitrogen of Asn167. In the "oxyanion hole", the amide carbonyl oxygen of the inhibitor interacts through a water molecule with the side chain of Asn167, the inhibitor's thiolate bridges the two Zn(II) ions in the active site displacing the bridging water, and the phenylbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 A compared to the unbound structure, allowing Trp28 to interact edge-to-face with the inhibitor's thiophene ring. The similarities between this inhibitor and the beta-lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo beta-lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the beta-lactam substrates. These interactions are complemented by additional nonspecific binding between the more variable groups in the substrates and the flexible flap. This unique mode of binding of the mercaptocarboxylate inhibitor in the enzyme active site provides a binding model for metallo beta-lactamase inhibition with utility for future drug design.