A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea)

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.     

X-ray and biochemical anatomy of an archaeal XPF/Rad1/Mus81 family nuclease: similarity between its endonuclease domain and restriction enzymes

The XPF/Rad1/Mus81-dependent nuclease family specifically cleaves branched structures generated during DNA repair, replication, and recombination, and is essential for maintaining genome stability. Here, we report the domain organization of an archaeal homolog (Hef) of this family and the X-ray crystal structure of the middle domain, with the nuclease activity. The nuclease domain architecture exhibits remarkable similarity to those of restriction endonucleases, including the correspondence of the GDX(n)ERKX(3)D signature motif in Hef to the PDX(n)(E/D)XK motif in restriction enzymes. This structural study also suggests that the XPF/Rad1/Mus81/ERCC1 proteins form a dimer through each interface of the nuclease domain and the helix-hairpin-helix domain. Simultaneous disruptions of both interfaces result in their dissociation into separate monomers, with strikingly reduced endonuclease activities.     

Studies on substrate recognition by the budding yeast separase

Sister chromatid cohesion is resolved at anaphase onset when separase, a site-specific protease, cleaves the Scc1 subunit of the chromosomal cohesin complex that is responsible for holding sister chromatids together. This mechanism to initiate anaphase is conserved in eukaryotes from budding yeast to man. Budding yeast separase recognizes and cleaves two conserved peptide motifs within Scc1. In addition, separase cleaves a similar motif in the kinetochore and spindle protein Slk19. Separase may cleave further substrate proteins to orchestrate multiple cellular events that take place during anaphase. To investigate substrate recognition by budding yeast separase we analyzed the sequence requirements at one of the Scc1 cleavage site motifs by systematic mutagenesis. We derived a cleavage site consensus motif (not(FKRWY))(ACFHILMPVWY)(DE)X(AGSV)R/X. This motif is found in 1,139 of 5,889 predicted yeast proteins. We analyzed 28 candidate proteins containing this motif as well as 35 proteins that contain a core (DE)XXR motif. We could so far not confirm new separase substrates, but we have uncovered other forms of mitotic regulation of some of the proteins. We studied whether determinants other than the cleavage site motif mediate separase-substrate interaction. When the separase active site was occupied with a peptide inhibitor covering the cleavage site motif, separase still efficiently interacted with its substrate Scc1. This suggests that separase recognizes both a cleavage site consensus sequence as well as features outside the cleavage site.     

3',5' Cyclic nucleotide phosphodiesterases class III: members, structure, and catalytic mechanism

3',5' Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that were previously divided by their primary structure into two major classes: PDE class I and II. The 3',5' cyclic AMP phosphodiesterase from Escherichia coli encoded by the cpdA gene does not show any homology to either PDE class I or class II enzymes and, therefore, represents a new, third class of PDEs. Previously, information about essential structural elements, substrate and cofactor binding sites, and the mechanism of catalysis was unknown for this enzyme. The present study shows by computational analysis that the enzyme encoded by the E. coli cpdA gene belongs to a family of phosphodiesterases that closely resembles the catalytic machinery known from purple acid phosphatases and several other dimetallophosphoesterases. They share both the conserved sequence motif, D-(X)(n) GD-(X)(n)-GNH[E/D]-(X)(n)-H-(X)(n)-GHXH, which contains the invariant residues forming the active site of purple acid phosphatases, a binuclear Fe(3+)-Me(2+)-containing center, as well as a beta(alpha)beta(alpha)beta motif as a typical secondary structure signature. Furthermore, the known biochemical properties of the bacterial phosphodiesterase encoded by the cpdA gene, such as the requirement of iron ions and a reductant for maintaining its catalytic activity, support this hypothesis developed by computational analysis. In addition, the availability of atomic coordinates for several purple acid phosphatases and related proteins allowed the generation of a three-dimensional model for class III cyclic nucleotide phosphodiesterases.     

Identification of mimotopes of the Japanese encephalitis virus envelope protein using phage-displayed combinatorial peptide library

The phage-displayed combinatorial peptide library is a revolutionary method for discovering epitopes, in particular conformational epitopes. In this study, we characterized a Japanese encephalitis virus (JEV) conformational epitope by biopanning of phage-displayed random peptide libraries with a JEV envelope (E) protein-specific monoclonal antibody (mAb) 2H2. Eleven identified phage clones with high affinity to mAb 2H2 were identified using direct and inhibitory binding ELISA. Sequence alignment, structure modeling and mutational analysis revealed that the identified mimotopes for mAb 2H2 possess a conserved motif X(1)(D/E)(Y/T/S)X(2), fitting into a region at the domain III lateral surface of the E protein. The results of our study could provide useful information on the development of effective mimotope-based vaccines and diagnostic kits for the JEV infection.     

Endonucleolytic function of MutLalpha in human mismatch repair

Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5'-to-3' activity of MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.     

X-ray structure of putative acyl-ACP desaturase DesA2 from Mycobacterium tuberculosis H37Rv

Genome sequencing showed that two proteins in Mycobacterium tuberculosis H37Rv contain the metal binding motif (D/E)X(2)HX(approximately 100)(D/E)X(2)H characteristic of the soluble diiron enzyme superfamily. These putative acyl-ACP desaturase genes desA1 and desA2 were cloned from genomic DNA and expressed in Escherichia coli BL21(DE3). DesA1 was found to be insoluble, but in contrast, DesA2 was a soluble protein amenable to biophysical characterization. Here, we report the 2.0 A resolution X-ray structure of DesA2 determined by multiple anomalous dispersion (MAD) phasing from a Se-met derivative and refinement against diffraction data obtained on the native protein. The X-ray structure shows that DesA2 is a homodimeric protein with a four-helix bundle core flanked by five additional helices that overlay with 192 structurally equivalent amino acids in the structure of stearoyl-ACP Delta9 desaturase from castor plant with an rms difference 1.42 A. In the DesA2 crystals, one metal (likely Mn from the crystallization buffer) was bound in high occupancy at the B-site of the conserved metal binding motif, while the A-site was not occupied by a metal ion. Instead, the amino group of Lys-76 occupied this position. The relationships between DesA2 and known diiron enzymes are discussed.     

Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis

M.EcoHK31I is a naturally occurring mC5-methyltransferase with a large alpha polypeptide and a small beta polypeptide. Polypeptide alpha contains conserved motifs I-VIII and X, and polypeptide beta contains motif IX. To understand how polypeptide alpha carries out its function, a molecular model of the large domain of polypeptide alpha was generated using M.HhaI and M.HaeIII as templates. The large domain is a mixed alpha/beta structure. Residues 15-19 in motif I (Phe-Naa-Gly-Naa) are conserved for cofactor binding. The key catalytic residue Cys-79 in motif IV is also conserved in comparison with other C-5 MTases. Comparing polypeptide alpha with M.HhaI and M.HaeIII revealed a unique region upstream of motif X. To understand the role of this region, 14 charged residues between R224 and E271 in the putative small domain were mutated. Activity assays indicated that most of these charges can be eliminated or changed conservatively. Among these charged residues, R224, E240, D245 and D251 may take part in proper interaction with DNA in the presence of polypeptide beta.     

Theoretical and experimental characterization of the scope of protein O-glycosylation in Bacteroides fragilis

Among bacterial species demonstrated to have protein O-glycosylation systems, that of Bacteroides fragilis and related species is unique in that extracytoplasmic proteins are glycosylated at serine or threonine residues within the specific three-amino acid motif D(S/T)(A/I/L/M/T/V). This feature allows for computational analysis of the proteome to identify candidate glycoproteins. With the criteria of a signal peptidase I or II cleavage site or a predicted transmembrane-spanning region and the presence of at least one glycosylation motif, we identified 1021 candidate glycoproteins of B. fragilis. In addition to the eight glycoproteins identified previously, we confirmed that another 12 candidate glycoproteins are in fact glycosylated. These included four glycoproteins that are predicted to localize to the inner membrane, a compartment not previously shown to include glycosylated proteins. In addition, we show that four proteins involved in cell division and chromosomal segregation, two of which are encoded by candidate essential genes, are glycosylated. To date, we have not identified any extracytoplasmic proteins containing a glycosylation motif that are not glycosylated. Therefore, based on the list of 1021 candidate glycoproteins, it is likely that hundreds of proteins, comprising more than half of the extracytoplasmic proteins of B. fragilis, are glycosylated. Site-directed mutagenesis of several glycoproteins demonstrated that all are glycosylated at the identified glycosylation motif. By engineering glycosylation motifs into a naturally unglycosylated protein, we are able to bring about site-specific glycosylation at the engineered sites, suggesting that this glycosylation system may have applications for glycoengineering.     

Association of potent human antiviral cytidine deaminases with 7SL RNA and viral RNP in HIV-1 virions

7SL RNA promotes the formation of the signal recognition particle that targets secretory and membrane proteins to the endoplasmic reticulum. 7SL RNA is also selectively packaged by many retroviruses, including HIV-1. Here, we demonstrate that 7SL RNA is an integral component of the viral ribonucleoprotein (RNP) complex containing Gag, viral genomic RNA, and tRNA(3)(Lys). Only the potent anti-HIV-1 cytidine deaminases can bind to 7SL RNA and target to HIV-1 RNP. A conserved motif in the amino-terminal region of A3G is important for 7SL RNA interaction. The weak anti-HIV-1 A3C did not interact with 7SL RNA and failed to target to viral RNPs, despite efficient virion packaging. However, a chimeric construct of A3C plus the 7SL-binding amino terminus of A3G did target to viral RNPs and showed enhanced anti-HIV-1 activity. 7SL RNA binding is a conserved feature of human anti-HIV-1 cytidine deaminases. Thus, potent anti-HIV-1 cytidine deaminases have evolved to possess a unique RNA-binding ability for precise HIV-1 targeting and viral inhibition.     

Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).     

A new definition for the consensus sequence of the peroxisome targeting signal type 2

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.     

Cytochrome b561 protein family: expanding roles and versatile transmembrane electron transfer abilities as predicted by a new classification system and protein sequence motif analyses

Cytochrome b561 family was characterized by the presence of "b561 core domain" that forms a transmembrane four helix bundle containing four totally conserved His residues, which might coordinate two heme b groups. We conducted BLAST and PSI-BLAST searches to obtain insights on structure and functions of this protein family. Analyses with CLUSTAL W on b561 sequences from various organisms showed that the members could be classified into 7 subfamilies based on characteristic motifs; groups A (animals/neuroendocrine), B (plants), C (insects), D (fungi), E (animals/TSF), F (plants+DoH), and G (SDR2). In group A, both motif 1, {FN(X)HP(X)2M(X)2G(X)5G(X)ALLVYR}, and motif 2, {YSLHSW(X)G}, were identified. These two motifs were also conserved in group B. There was no significant features characteristic to groups C and D. A modified version of motif 1, {LFSWHP(X)2M(X)3F(X)3M(X)EAIL(X)SP(X)2SS}, was found in group E with a high degree of conservation. Both motif 3, {DP(X)WFY(L)H(X)3Q}, and motif 4, {K(X)R(X)YWN(X)YHH(X)2G(R/Y)} ,were found in group F at different regions from those of motifs 1 and 2. The "DoH" domain common to the NH2-terminal region of dopamine beta-hydroxylase was found to form fusion proteins with the b561 core domains in groups F and G. Based on these results, we proposed a hypothesis regarding structures and functions of the 7 subfamilies of cytochrome b561.     

3D-QSAR study for DNA cleavage proteins with a potential anti-tumor ATCUN-like motif

Genomics projects have elucidated several genes that encode protein sequences. Subsequently, the advent of the proteomics age has enabled the synthesis and 3D structure determination for these protein sequences. Some of these proteins incorporate metal atoms but it is often not known whether they are metal-binding proteins and the nature of the biological activity is not understood. Consequently, the development of methods to predict metal-mediated biological activity of proteins from the 3D structure of metal-unbound proteins is a goal of major importance. More specifically, the amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif is a small metal-binding site found in the N-terminus of many naturally occurring proteins. The ATCUN motif participates in DNA cleavage and has anti-tumor activity. In this study, we calculated average 3D electrostatic potentials (xi(k)) for 265 different proteins including 133 potential ATCUN anti-tumor proteins. We also calculated xi(k) values for the total protein or for the following specific protein regions: the core, inner, middle, and outer orbits. A linear discriminant analysis model was subsequently developed to assign proteins into two groups called ATCUN DNA-cleavage proteins and non-active proteins. The best model found was: ATCUN=1.15.xi(1)(inner)+2.18.xi(5)(middle)+27.57.xi(0)(outer)-27.57.xi(0)(total)+0.09. The model correctly classified 182 out of 197 (91.4%) and 61 out of 66 (92.4%) proteins in training and external predicting series', respectively. Finally, desirability analysis was used to predict the values for the electrostatic potential in one single region and the combined values in two regions that are desirable for ATCUN-like proteins. To the best of our knowledge, the present work is the first study in which desirability analysis has been used in protein quantitative-structure-activity-relationship (QSAR).     

Multistep binding of transition metals to the H-N-H endonuclease toxin colicin E9

We report the first stopped-flow fluorescence analysis of transition metal binding (Co(2+), Ni(2+), Cu(2+), and Zn(2+)) to the H-N-H endonuclease motif within colicin E9 (the E9 DNase). The H-N-H consensus forms the active site core of a number of endonuclease groups but is also structurally homologous to the so-called treble-clef motif, a ubiquitous zinc-binding motif found in a wide variety of metalloproteins. We find that all the transition metal ions tested bind via multistep mechanisms. Binding was further dissected for Ni(2+) and Zn(2+) ions through the use of E9 DNase single tryptophan mutants, which demonstrated that most steps reflect conformational rearrangements that occur after the bimolecular collision, many common to the two metals, while one appears specific to zinc. The kinetically derived equilibrium dissociation constants (K(d)) for transition metal binding to the E9 DNase agree with previously determined equilibrium measurements and so confirm the validity of the derived kinetic mechanisms. Zn(2+) binds tightest to the enzyme (K(d) approximately 10(-)(9) M) but does not support endonuclease activity, whereas the other metals (K(d) approximately 10(-)(6) M) are active in endonuclease assays implying that the additional step seen for Zn(2+) traps the enzyme in an inactive but high affinity state. Metal-induced conformational changes are likely to be a conserved feature of H-N-H/treble clef motif proteins since similar Zn(2+)-induced, multistep binding was observed for other colicin DNases. Moreover, they appear to be independent both of the conformational heterogeneity that is naturally present within the E9 DNase at equilibrium, as well as the conformational changes that accompany the binding of its cognate inhibitor protein Im9.     

Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional Gbeta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy

The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000) Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser(879) and Ser(880), are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gbeta-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade.     

Definition of the consensus motif recognized by gamma-adaptin ear domains

The heterotetrameric adaptor complex 1 (AP-1) and the monomeric Golgi-localized, gamma ear-containing, Arf-binding (GGA) proteins are components of clathrin coats associated with the trans-Golgi network and endosomes. The carboxyl-terminal ear domains (or gamma-adaptin ear (GAE) domains) of two gamma-adaptin subunit isoforms of AP-1 and of the GGAs are structurally similar and bind to a common set of accessory proteins. In this study, we have systematically defined a core tetrapeptide motif PsiG(P/D/E)(Psi/L/M) (where Psi is an aromatic residue), which is responsible for the interactions of accessory proteins with GAE domains. The definition of this motif has allowed us to identify novel GAE-binding partners named NECAP and aftiphilin, which also contain clathrin-binding motifs. These findings shed light on the mechanism of accessory protein recruitment to trans-Golgi network and endosomal clathrin coats.     

Rigidity and flexibility characteristics of DD[E/D]-transposases Mos1 and Sleeping Beauty

DD[E/D]-transposases catalyze the multistep reaction of cut-and-paste DNA transposition. Structurally, several DD[E/D]-transposases have been characterized, revealing a multi-domain structure with the catalytic domain possessing the RNase H-like structural motif that brings three catalytic residues (D, D, and E or D) into close proximity for the catalysis. However, the dynamic behavior of DD[E/D]-transposases during transposition remains poorly understood. Here, we analyze the rigidity and flexibility characteristics of two representative DD[E/D]-transposases Mos1 and Sleeping Beauty (SB) using the minimal distance constraint model (mDCM). We find that the catalytic domain of both transposases is globally rigid, with the notable exception of the clamp loop being flexible in the DNA-unbound form. Within this globally rigid structure, the central β-sheet of the RNase H-like motif is much less rigid in comparison to its surrounding α-helices, forming a cage-like structure. The comparison of the original SB transposase to its hyperactive version SB100X reveals the region where the change in flexibility/rigidity correlates with increased activity. This region is found to be within the RNase H-like structural motif and comprise the loop leading from beta-strand B3 to helix H1, helices H1 and H2, which are located on the same side of the central beta-sheet, and the loop between helix H3 and beta-strand B5. We further identify the RKEN214-217DAVQ mutations of the set of hyperactive mutations within the catalytic domain of SB transposase to be the driving factor that induces change in residue-pair rigidity correlations within SB transposase. Given that a signature RNase H-like structural motif is found in DD[E/D]-transposases and, more broadly, in a large superfamily of polynucleotidyl transferases, our results are relevant to these proteins as well.     

Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome.

Protein phosphatase 1 (Glc7p) and its binding protein Reg1p are essential for the regulation of glucose repression pathways in Saccharomyces cerevisiae. In order to identify physiological substrates for the Glc7p-Reg1p complex, we examined the effects of deletion of the REG1 gene on the yeast phosphoproteome. Analysis by two-dimensional phosphoprotein mapping identified two distinct proteins that were greatly increased in phosphate content in reg1Delta mutants. Mixed peptide sequencing identified these proteins as hexokinase II (Hxk2p) and the E1alpha subunit of pyruvate dehydrogenase. Consistent with increased phosphorylation of Hxk2p in response to REG1 deletion, fractionation of yeast extracts by anion-exchange chromatography identified Hxk2p phosphatase activity in wild-type strains that was selectively lost in the reg1Delta mutant. The phosphorylation state of Hxk2p and Hxk2p phosphatase activity was restored to wild-type levels in the reg1Delta mutant by expression of a LexA-Reg1p fusion protein. In contrast, expression of LexA-Reg1p containing mutations at phenylalanine in the putative PP-1C-binding site motif (K/R)(X)(I/V)XF was unable to rescue Hxk2p dephosphorylation in intact yeast or restore Hxk2p phosphatase activity. These results demonstrate that Reg1p targets PP-1C to dephosphorylate Hxk2p in vivo and that the motif (K/R)(X) (I/V)XF is necessary for its PP-1 targeting function.