Specificity and affinity of sialic acid binding by the rhesus rotavirus VP8* core

Nuclear magnetic resonance spectroscopy demonstrates that the rhesus rotavirus hemagglutinin specifically binds alpha-anomeric N-acetylneuraminic acid with a K(d) of 1.2 mM. The hemagglutinin requires no additional carbohydrate moieties for binding, does not distinguish 3' from 6' sialyllactose, and has approximately tenfold lower affinity for N-glycolylneuraminic than for N-acetylneuraminic acid. The broad specificity and low affinity of sialic acid binding by the rotavirus hemagglutinin are consistent with this interaction mediating initial cell attachment prior to the interactions that determine host range and cell type specificity.     

The Gla domain of human prothrombin has a binding site for factor Va

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.     

Modeling a sialic acid binding pocket in the external loops of JC virus VP1

JC virus (JCV) is a common human polyomavirus that infects over 70% of the population worldwide. JCV has a restricted cell tropism that is caused partly by the initial interaction between the virus and sialic acid-containing host cell receptors. To identify the molecular interactions between the virus and its cellular receptor, we used a combined approach of site-directed mutagenesis and homology-based molecular modeling. A model of the major viral capsid protein VP1 based on sequence alignment with other closely related polyomaviruses allowed us to target specific amino acids in the extracellular loops of VP1 for mutagenesis. An analysis of the growth rates of 17 point mutants led to the identification of VP1 amino acids that are critical in virus-host cell receptor interactions. Molecular dynamics simulations were then used to build and confirm a model of the interaction between VP1 and the sialic acid component of the JCV receptor.     

Mycobacterium tuberculosis ribose-5-phosphate isomerase has a known fold, but a novel active site

Ribose-5-phosphate isomerases (EC 5.3.1.6) inter-convert ribose-5-phosphate and ribulose-5-phosphate. This reaction allows the synthesis of ribose from other sugars, as well a means for salvage of carbohydrates after nucleotide breakdown. Two unrelated types of enzyme are known to catalyze the isomerization. The most common one, RpiA, is present in almost all organisms. The second type, RpiB, is found in many bacterial species.Here, we demonstrate that the RpiB from Mycobacterium tuberculosis (Rv2465c) has catalytic properties very similar to those previously reported for the Escherichia coli RpiB enzyme. Further, we report the structure of the mycobacterial enzyme, solved by molecular replacement and refined to 1.88A resolution. Comparison with the E.coli structure shows that there are important differences in the two active sites, including a change in the position and nature of the catalytic base. Sequence comparisons reveal that the M.tuberculosis and E.coli RpiB enzymes are in fact representative of two distinct sub-families. The mycobacterial enzyme represents a type found only in actinobacteria, while the enzyme from E.coli is typical of that seen in many other bacterial proteomes. Both RpiBs are very different from RpiA in structure as well as in the construction of the active site. Docking studies allow additional insights into the reactions of all three enzymes, and show that many features of the mechanism are preserved despite the different catalytic components.     

The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site

ATP-dependent Lon protease degrades specific short-lived regulatory proteins as well as defective and abnormal proteins in the cell. The crystal structure of the proteolytic domain (P domain) of the Escherichia coli Lon has been solved by single-wavelength anomalous dispersion and refined at 1.75-A resolution. The P domain was obtained by chymotrypsin digestion of the full-length, proteolytically inactive Lon mutant (S679A) or by expression of a recombinant construct encoding only this domain. The P domain has a unique fold and assembles into hexameric rings that likely mimic the oligomerization state of the holoenzyme. The hexamer is dome-shaped, with the six N termini oriented toward the narrower ring surface, which is thus identified as the interface with the ATPase domain in full-length Lon. The catalytic sites lie in a shallow concavity on the wider distal surface of the hexameric ring and are connected to the proximal surface by a narrow axial channel with a diameter of approximately 18 A. Within the active site, the proximity of Lys(722) to the side chain of the mutated Ala(679) and the absence of other potential catalytic side chains establish that Lon employs a Ser(679)-Lys(722) dyad for catalysis. Alignment of the P domain catalytic pocket with those of several Ser-Lys dyad peptide hydrolases provides a model of substrate binding, suggesting that polypeptides are oriented in the Lon active site to allow nucleophilic attack by the serine hydroxyl on the si-face of the peptide bond.     

STD NMR spectroscopy and molecular modeling investigation of the binding of N-acetylneuraminic acid derivatives to rhesus rotavirus VP8* core

The VP8* subunit of rotavirus spike protein VP4 contains a sialic acid (Sia)-binding domain important for host cell attachment and infection. In this study, the binding epitope of the N-acetylneuraminic acid (Neu5Ac) derivatives has been characterized by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. From this STD NMR data, it is proposed that the VP8* core recognizes an identical binding epitope in both methyl alpha-D-N-acetylneuraminide (Neu5Acalpha2Me) and the disaccharide methyl S-(alpha-D-N-acetylneuraminosyl)-(2-->6)-6-thio-beta-D-galactopyranoside (Neu5Ac-alpha(2,6)-S-Galbeta1Me). In the VP8*-disaccharide complex, the Neu5Ac moiety contributes to the majority of interaction with the protein, whereas the galactose moiety is solvent-exposed. Molecular dynamics calculations of the VP8*-disaccharide complex indicated that the galactose moiety is unable to adopt a conformation that is in close proximity to the protein surface. STD NMR experiments with methyl 9-O-acetyl-alpha-D-N-acetylneuraminide (Neu5,9Ac(2)alpha2Me) in complex with rhesus rotavirus (RRV) VP8* revealed that both the N-acetamide and 9-O-acetate moieties are in close proximity to the Sia-binding domain, with the N-acetamide's methyl group being saturated to a larger extent, indicating a closer association with the protein. RRV VP8* does not appear to significantly recognize the unsaturated Neu5Ac derivative [2-deoxy-2,3-didehydro-D-N-acetylneuraminic acid (Neu5Ac2en)]. Molecular modeling of the protein-Neu5Ac2en complex indicates that key interactions between the protein and the unsaturated Neu5Ac derivative when compared with Neu5Acalpha2Me would not be sustained. Neu5Acalpha2Me, Neu5Ac-alpha(2,6)-S-Galbeta1Me, Neu5,9Ac(2)alpha2Me, and Neu5Ac2en inhibited rotavirus infection of MA104 cells by 61%, 35%, 30%, and 0%, respectively, at 10 mM concentration. NMR spectroscopic, molecular modeling, and infectivity inhibition results are in excellent agreement and provide valuable information for the design of inhibitors of rotavirus infection.     

The rhesus rotavirus gene encoding VP4 is a major determinant in the pathogenesis of biliary atresia in newborn mice

Biliary atresia (BA) is a devastating disease of childhood for which increasing evidence supports a viral component in pathogenesis. The murine model of BA is induced by perinatal infection with rhesus rotavirus (RRV) but not with other strains of rotavirus, such as TUCH. To determine which RRV gene segment(s) is responsible for pathogenesis, we used the RRV and TUCH strains to generate a complete set of single-gene reassortants. Eleven single-gene "loss-of-function" reassortants in which a TUCH gene replaced its RRV equivalent and 11 single-gene "gain-of-function" reassortants in which an RRV gene replaced its TUCH equivalent were generated. Newborn BALB/c mice were inoculated with the reassortants and were monitored for biliary obstruction and mortality. In vitro, the ability to bind to and replicate within cholangiocytes was analyzed. Infection of mice with the "loss-of-function" reassortant R(T(VP4)), where gene 4 from TUCH was placed on an RRV background, eliminated the ability of RRV to cause murine BA. In a reciprocal fashion, the "gain-of-function" reassortant T(R(VP4)) resulted in murine BA with 88% mortality. Compared with those for RRV, R(T(VP4)) binding and titers in cholangiocytes were significantly attenuated, while T(R(VP4)) binding and titers were significantly increased over those for TUCH. Reassortants R(T(VP3)) and T(R(VP3)) induced an intermediate phenotype. RRV gene segment 4 plays a significant role in governing tropism for the cholangiocyte and the ability to induce murine BA. Gene segment 3 did not affect RRV infectivity in vitro but altered its in vivo effect.     

Initial interaction of rotavirus strains with N-acetylneuraminic (sialic) acid residues on the cell surface correlates with VP4 genotype, not species of origin

We examined 41 human and animal rotavirus strains representative of all known P genotypes for their dependency on cellular N-acetylneuraminic (sialic) acid (SA) residues for infectivity. Our results showed that all rotaviruses studied, whether of animal or human origin, belonging to P genotypes [1], [2], [3], and [7] depended on SA residues on the cell surface for efficient infectivity but that all human and animal rotavirus strains representative of the remaining known P genotypes were SA independent. The SA residue requirement for efficient infectivity did not change for reassortant rotavirus strains with altered VP4-VP7 combinations. The initial interaction of rotavirus strains with SA residues on the cell surface correlated with VP4 genotype specificity, not with species of origin or VP7 G serotype specificity (P = 0.001; r2 = 1.00, Pearson's correlation coefficient). In addition to being a requirement for infectivity, the presence of SA residues on the cell surface is a requirement for efficient growth in cell culture; recognition of the association of specific P genotypes with the binding of rotavirus to SA residues will facilitate our understanding of the molecular basis of the early events of rotavirus-cell interactions in cell culture models and of pathogenicity in vivo.     

Complete nucleotide sequence of the gene encoding VP4 of a human rotavirus (strain K8) which has unique VP4 neutralization epitopes.

In our previous study (K. Taniguchi, Y. Morita, T. Urasawa, and S. Urasawa, J. Virol. 62:2421-2426, 1987) in which the cross-reactive neutralization epitopes on VP4 of human rotaviruses were analyzed, one strain, K8, was found to bear unique VP4 neutralization epitopes. This strain, which belongs to subgroup II and serotype 1, was not neutralized by any of six anti-VP4 neutralizing monoclonal antibodies which reacted with human rotavirus strains of serotypes 1, 3, and 4 or serotypes 1 through 4. We determined the complete nucleotide sequence of the gene encoding VP4 of strain K8 by primer extension. The VP4 gene is 2,359 base pairs in length, with 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. The gene contains a long open reading frame of 2,325 bases capable of coding for a protein of 775 amino acids. When compared with those of other human rotaviruses, VP4 of strain K8 had an insertion of one amino acid after residue 135, as found in simian rotavirus strains, and in addition, it had a deletion of one amino acid (residue 575). The amino acid homology of VP4 of strain K8 and those of other virulent human rotaviruses was only 60 to 70%. This was unusual, since over 90% VP4 homology has been found among the other virulent human rotavirus strains. In contrast, the VP7 amino acid sequence of the K8 strain was quite similar (over 98% homology) to those of other serotype 1 human rotaviruses. Thus, the K8 strain appears to have a unique VP4 gene previously not described.     

An unusual carbohydrate binding site revealed by the structures of two Maackia amurensis lectins complexed with sialic acid-containing oligosaccharides

Seeds from the legume tree Maackia amurensis contain two lectins that can agglutinate different blood cell types. Their specificity toward sialylated oligosaccharides is unique among legume lectins; the leukoagglutinin preferentially binds to sialyllactosamine (alphaNeuAc(2-3)betaGal(1-4)betaGlcNAc), whereas the hemagglutinin displays higher affinity for a disialylated tetrasaccharide (alphaNeuAc(2-3)betaGal(1-3)[alphaNeuAc(2-6)]alphaG alNAc). The three-dimensional structure of the complex between M. amurensis leukoagglutinin and sialyllactose has been determined at 2.75-A resolution using x-ray crystallography. The carbohydrate binding site consists of a deep cleft that accommodates the three carbohydrate residues of the sialyllactose. The central galactose sits in the primary binding site in an orientation that has not been observed previously in other legume lectins. The carboxyl group of sialic acid establishes a salt bridge with a lysine side chain. The glucose residue is very efficiently docked between two tyrosine aromatic rings. The complex between M. amurensis hemagglutinin and a disialylated tetrasaccharide could be modeled from the leukoagglutinin/sialyllactose crystal structure. The substitution of one tyrosine by an alanine residue is responsible for the difference in fine specificity between the two isolectins. Comparison with other legume lectins indicates that oligosaccharide specificity within this family is achieved by the recycling of structural loops in different combinations.     

2009～2010年北京儿童腹泻中轮状病毒新VP4基因亚型的研究

P[8]b基因亚型是国内外新近发现的A组人轮状病毒(HRV)VP4基因的一种新亚型,本研究旨在建立有效鉴别HRV P[8]a和P[8]b基因亚型及P[4]和P[6]基因型的VP4基因打点杂交分型方法,并运用此方法对2009～2010年首都儿科研究所附属儿童医院门诊及住院腹泻患儿中P[8]b基因亚型HRV的流行情况及其G/P基因组合情况进行研究。通过对GenBank序列数据库可检索到的国内外HRV各种P基因型及亚型的VP4基因序列应用相关软件进行基因分析,在不同基因型别间核苷酸变异密集而相同P基因型内核苷酸高度保守的的位置设计各型别探针,并分别以本实验室上传GenBank的北京HRV地方株P[4]和P[6]基因型及P[8]a和P[8]b亚型的VP4基因作为相应型别探针的合成引物的设计模板及探针经PCR合成的合成模板,合成地高辛素标记的DNA探针。经测序验证所建立的VP4基因杂交分型方法结果可靠。对门诊88例(55%,88/160)及住院79例(70.5%,79/112)HRV腹泻患儿的P分型结果显示P[8]a亚型仍为主要型别,前者为96.6%(85/88),而后者为62.0%(49/79);P[8]b亚型在住院HRV感染腹泻患儿中占较高比例(27.9%,22/79),虽然其在门诊HRV感染患儿中也存在,但仅占2.3%(2/88);另外单纯P[4]基因型HRV感染仅在住院腹泻患儿中检测到1例(1.3%,1/79),而P[6]基因型在门诊及住院HRV感染腹泻患儿中均未检测到;本组标本中HRV P[8]b亚型主要与G9基因型组合。本研究表明G9P[8]b型HRV在北京腹泻儿童中有流行。     

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site

The fibronectin-binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μM). The binding site for FnBPB in fibrinogen was localized to the C-terminus of the γ-chain. Like clumping factor A, region A of FnBPB bound to the γ-chain of fibrinogen in a Ca(2+)-inhibitable manner. The deletion of 17 residues from the C-terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock-lock-latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with K(D) = 2.5 μM despite lacking any of the known fibronectin-binding tandem repeats. A truncate lacking the C-terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, K(D) of 20 μM, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin-binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.     

Crystal structure of the oligomerization domain of NSP4 from rotavirus reveals a core metal-binding site

During the maturation of rotaviral particles, non-structural protein 4 (NSP4) plays a critical role in the translocation of the immature capsid into the lumen of the endoplasmic reticulum. Full-length NSP4 and a 22 amino acid peptide (NSP4(114-135)) derived from this protein have been shown to induce diarrhea in young mice in an age-dependent manner, and may therefore be the agent responsible for rotavirally-induced symptoms. We have determined the crystal structure of the oligomerization domain of NSP4 which spans residues 95 to 137 (NSP4(95-137)). NSP4(95-137) self-associates into a parallel, tetrameric coiled-coil, with the hydrophobic core interrupted by three polar layers occupying a and d-heptad positions. Side-chains from two consecutive polar layers, consisting of four Gln123 and two of the four Glu120 residues, coordinate a divalent cation. Two independent structures built from MAD-phased data indicated the presence of a strontium and calcium ion bound at this site, respectively. This metal-binding site appears to play an important role in stabilizing the homo-tetramer, which has implications for the engagement of NSP4 as an enterotoxin.     

Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site

The grass Lolium perenne produces an ice-binding protein (LpIBP) that helps this perennial tolerate freezing by inhibiting the recrystallization of ice. Ice-binding proteins (IBPs) are also produced by freeze-avoiding organisms to halt the growth of ice and are better known as antifreeze proteins (AFPs). To examine the structural basis for the different roles of these two IBP types, we have solved the first crystal structure of a plant IBP. The 118-residue LpIBP folds as a novel left-handed beta-roll with eight 14- or 15-residue coils and is stabilized by a small hydrophobic core and two internal Asn ladders. The ice-binding site (IBS) is formed by a flat beta-sheet on one surface of the beta-roll. We show that LpIBP binds to both the basal and primary-prism planes of ice, which is the hallmark of hyperactive AFPs. However, the antifreeze activity of LpIBP is less than 10% of that measured for those hyperactive AFPs with convergently evolved beta-solenoid structures. Whereas these hyperactive AFPs have two rows of aligned Thr residues on their IBS, the equivalent arrays in LpIBP are populated by a mixture of Thr, Ser and Val with several side-chain conformations. Substitution of Ser or Val for Thr on the IBS of a hyperactive AFP reduced its antifreeze activity. LpIBP may have evolved an IBS that has low antifreeze activity to avoid damage from rapid ice growth that occurs when temperatures exceed the capacity of AFPs to block ice growth while retaining the ability to inhibit ice recrystallization.     

The Geodia cydonium galectin exhibits prototype and chimera-type characteristics and a unique sequence polymorphism within its carbohydrate recognition domain

The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAcalpha1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing.     

The RRM domain of human fused in sarcoma protein reveals a non-canonical nucleic acid binding site

Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS.     

The PX domain as a novel phosphoinositide- binding module

The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox), the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: p40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P(2). In addition, the Bem1p PX domain also interacts with Ptdlns(4)P. When the p40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant p40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes.     

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.