Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases. Comparison with human liver L-type enzyme

Human red cell contain soluble adenosine-3',5'-phosphate-dependent protein kinases, which are able to phosphorylate the L' subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L'4 enzyme and accounts for less than 20% of total pyruvate kinase subunits. Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.     

The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.     

Phosphorylase kinase specificity. A comparative study with cAMP-dependent protein kinase on synthetic peptides and peptide analogs of glycogen synthase and phosphorylase

A synthetic pentadecapeptide, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ser-Ser-Leu-Pro-Gly-Leu-Glu, corresponding to the phosphorylatable site at the NH2 terminus of glycogen synthase, could be phosphorylated stoichiometrically at seryl residue 7 by both phosphorylase kinase and cAMP-dependent protein kinase. Phosphorylation of seryl residue 3 also occurred after prolonged incubation with cAMP-dependent protein kinase. Kinetic studies show that the pentadecapeptide is a better substrate for phosphorylase kinase. A peptide consisting of residues 1-11 was not as good a substrate and substitution of Arg-4 by Lys and Ser-9 by ARg in the unidecapeptide decreased and increased phosphorylase kinase reaction rates, respectively. Higher rates of phosphorylation were obtained with peptides of the phosphorylatable site of phosphorylase. A peptide with the sequence, Leu-Ser-Tyr-Arg-Arg-Tyr-Ser-Leu was phosphorylated initially by phosphorylase kinase and cAMP-dependent protein kinase at Ser-2 and Ser-7, respectively. Upon longer incubation, second site phosphorylation occurred with both kinases. A peptide of the same sequence with D-amino acids could not be phosphorylated but was a competitive inhibitor of both enzymes. The results suggest that optimal interaction of the two kinases depends on various factors including the orientation of arginyl groups with respect to the phosphorylatable serine.     

Identification of three phosphorylation sites in the alpha7 subunit of the yeast 20S proteasome in vivo using mass spectrometry

The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.     

Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates

The factors determining the site recognition and phosphorylation by rat liver casein kinase-2 (CK-2) have been explored with a set of 14 related hexapeptides each including a single phosphorylatable amino acid and five acidic plus neutral residues. Such peptides are different from each other in the following features: the nature of the phosphorylatable amino acid, if any; its position relative to the critically required acidic residues; the extension and the structure of the acidic cluster. All of them were tested as substrate and/or competitive inhibitors of CK-2, and their kinetic and inhibition constants were determined. The results suggest the following conclusions. Under strictly comparable conditions Ser is by far preferred over Thr. Tyr not being affected at all. In order to carry out its role of structural determinant the critical acidic cluster must be located on the C-terminal side of the target residue, though not necessarily adjacent to it. The affinity for the protein-binding site, as deduced from Km and/or Ki values, is largely dependent on the number of acidic residues but it is also significantly enhanced if a hydroxylic residue is located on their N-terminal side. An acidic residue at position +3 relative to serine plays an especially important role for triggering phosphorylation, the peptide Ser-Glu-Glu-Ala-Glu-Glu having similar Km but negligible Vmax compared to Ser-Glu-Ala-Glu-Glu-Glu and Ser-Glu-Glu-Glu-Ala-Glu. These data provide a rationale for the substrate specificity of CK-2 and will give a helpful insight into the structure of the protein-binding site of this enzyme.     

Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase

Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.     

Studies of the phosphorylation of Escherichia coli isocitrate dehydrogenase. Recognition of the enzyme by isocitrate dehydrogenase kinase/phosphatase and effects of phosphorylation on its structure and properties

Escherichia coli isocitrate dehydrogenase is completely inactivated by phosphorylation of a single serine residue per subunit. We have examined the conformations of the active and phosphorylated forms of the enzyme using circular dichroism spectroscopy. The results support the view that phosphorylation prevents the binding of NADP, probably by direct blocking of the coenzyme-binding site. Labelling studies suggest that an arginine residue at the coenzyme-binding site may be close to the phosphorylatable serine residue. The phosphorylation of isocitrate dehydrogenase is thus unusual in that it occurs at the active site of the enzyme. We therefore investigated the recognition of isocitrate dehydrogenase by isocitrate dehydrogenase kinase/phosphatase. The kinase activity of this enzyme can phosphorylate intact isocitrate dehydrogenase but not proteolytic fragments derived from it, nor a synthetic peptide corresponding to the sequence round the phosphorylation site.     

Synthetic peptide substrates for casein kinase 2. Assessment of minimum structural requirements for phosphorylation

Unlike the peptides SAEAAA and SEEAAA which are not substrates for casein kinase 2 (CK-2) their analogs SAAEAE and SAAEAA are still significantly phosphorylated. Their K_m values, however, (13.3 and 18.9 mM, respectively) are almost two orders of magnitude higher than that of SEEEEE and their V_max values are 3- and 14-fold lower than that of SAAEEE. The peptide ESEEEEE, but not ASEEEEE, is a slightly better substrate than SEEEEE, while both RSEEEEE and SEEEKE are very poor substrates compared to ASEEEEE and SEEEAE, respectively. SAAEAE is much more responsive to polylysine stimulation and polyphosphate inhibition than is SEEEEE. Taken together these data show that a single acidic residue at the third position from the C-terminal side of the phosphorylatable amino acid represents not only a necessary, but also a sufficient condition for site recognition by CK-2. Optimal phosphorylation efficiency, however, requires an extended C-terminal cluster of several acidic residues, and can be compromised by the presence of only a basic residue either inside the acidic cluster or adjacent to the N-terminal side of the phosphoacceptor amino acid. The structure of the phosphoacceptor site can greatly influence the efficacy of substrate-directed effectors of CK-2.     

DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a major endogenous cytosolic substrate for dopamine- and cyclic AMP-stimulated protein phosphorylation in neurons of the basal ganglia of mammalian brain. It shares many properties with phosphatase inhibitor 1, a substrate for cyclic AMP-dependent protein kinase, and with G-substrate, a substrate for cyclic GMP-dependent protein kinase. We have, therefore, undertaken an analysis of the amino acid sequence around the site at which purified DARPP-32 is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. The results indicate that DARPP-32 is phosphorylated at a single threonine residue contained in the sequence Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Met-Leu-Phe-Arg. This sequence was obtained by automated solid phase sequencing of two overlapping tryptic phosphopeptides and one overlapping chymotryptic phosphopeptide which were purified by reverse-phase high-performance liquid chromatography. A 9-amino acid sequence containing the phosphorylatable threonine residue in DARPP-32 shares 8 identical residues with a sequence containing the phosphorylatable threonine residue in phosphatase inhibitor 1, and shares 5 identical residues with the two identical sequences surrounding the 2 phosphorylatable threonine residues in G-substrate. These observations support the view that DARPP-32, inhibitor 1, and G-substrate are members of a family of regulatory proteins which are involved in the control of protein phosphatase activity by both cyclic AMP and cyclic GMP, but which differ in their cellular and tissue distributions.     

Cloning of uvrA, lexC and ssb genes of Escherichia coli.

The L' subunits, phosphorylatable precursors of L-type pyruvate kinase, can be proteolyzed in vivo and in vitro, and transformed into several molecular species.Trypsin induces the appearance of a phosphorylatable form similar to liver L-type enzyme.Subtilisin splits the phosphorylatable site and induces the appearance of a form similar to the minor form occuring with red cell aging.Sensitivity to proteolysis of some pyruvate kinase variants is modified.     

Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.

The intact, 100 kd microsomal enzyme and the 53 kd catalytic fragment of rat HMG-CoA reductase are both phosphorylated and inactivated by the AMP-activated protein kinase. Using the catalytic fragment, we have purified and sequenced peptides containing the single site of phosphorylation. Comparison with the amino acid sequence predicted from the cDNAs encoding other mammalian HMG-CoA reductases identifies this site as a serine residue close to the C-terminus (Ser872 in the human enzyme). Phosphopeptide mapping of native, 100 kd microsomal HMG-CoA reductase confirms that this C-terminal serine is the only major site phosphorylated in the intact enzyme by the AMP-activated protein kinase. The catalytic fragment of HMG-CoA reductase was also isolated from rat liver in the presence of protein phosphatase inhibitors under conditions where the enzyme is largely in the inactive form. HPLC, mass spectrometry and sequencing of the peptide containing Ser872 demonstrated that this site is highly phosphorylated in intact liver under these conditions. We have also identified by amino acid sequencing the N-terminus of the catalytic fragment, which corresponds to residue 423 of the human enzyme.     

Phosphorylation of synthetic peptides by (32P)ATP and cyclic GMP-stimulated protein kinase.

Cyclic GMP-stimulated protein kinase from pig lung has been shown to phosphorylate synthetic peptides. The rate of phosphorylation was about one order of magnitude higher than that for mixed histones at a comparable concentration, $\text{i.e.}$ 0.1 mM. The peptides represented sites, phosphorylatable by cyclic AMP-stimulated protein kinase, in pyruvate kinase type L from rat liver, calf thymus histone H2B and the α-subunit of rabbit muscle phosphorylase b kinase. The shortest pyruvate kinase peptide that could be phosphorylated at a significant rate by cyclic GMP-stimulated protein kinase was Arg-Arg-Ala-Ser-Val-Ala, which is one amino acid residue longer than the minimal substrate of cyclic AMP-stimulated protein kinase. The apparent K_m was 0.3 mM which is about 10 times higher than that with cyclic AMP-stimulated protein kinase. The K_m was only slightly decreased upon successive extension of the peptide in the N-terminal direction to Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala. Modification of the sequence showed the importance of two adjacent arginyl residues, and substitution of arginine for the C-terminal alanine abolished the measurable activity. Thus, it has been demonstrated that there are both differences and similarities in substrate specificity of the two protein kinases.     

Specific Ser-Pro phosphorylation by the RNA-recognition motif containing kinase KIS.

We present here a first appraisal of the phosphorylation site specificity of KIS (for 'kinase interacting with stathmin'), a novel mammalian kinase that has the unique feature among kinases to possess an RNP type RNA-recognition motif (RRM). In vitro kinase assays using various standard substrates revealed that KIS has a narrow specificity, with myelin basic protein (MBP) and synapsin I being the best in vitro substrates among those tested. Mass spectrometry and peptide sequencing allowed us to identify serine 164 of MBP as the unique site phosphorylated by KIS. Phosphorylation of synthetic peptides indicated the importance of the proline residue at position +1. We also identified a tryptic peptide of synapsin I phosphorylated by KIS and containing a phosphorylatable Ser-Pro motif. Altogether, our results suggest that KIS preferentially phosphorylates proline directed residues but has a specificity different from that of MAP kinases and cdks.     

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.     

Quaternary structure and oxygenase activity of D-ribulose-1,5-bisphosphate carboxylase from Hydrogenomonas eutropha.

Electrophoretically homogeneous ribulose-1,5-bisphosphate (RuBP) carboxylase was obtained from autotropically grown Hydrogenomonas eutropha by sedimentation of the 105,000 X g supernatant in a discontinuous sucrose gradient and by ammonium sulfate fractionation followed by another sucrose gradient centrifugation. The molecular weight of the enzyme determined by light scattering was 490,000 +/- 15,000. The enzyme could be dissociated by sodium dodecyl sulfate into three types of subunits, and the molecular weights (+/- 10%) could be measured. There were two species of large subunits, L and L' (molecular weight 56,000 and 52,000, respectively) and one species of small subunits (molecular weight, 15,000). The mole ratio of L to L' was 5:3, and the overall mole ratio of the small to large subunits was 1.08. The simplest quaternary structure of the enzyme is L5L'3S8. The enzyme contained RuBP oxygenase activity as evidenced by the O2-dependent production of phosphoglycolate and 3-phosphoglyceric acid in equimolar quantities from RuBP.     

The minimum substrate of cyclic AMP-stimulated protein kinase, as studied by synthetic peptides representing the phosphorylatable site of pyruvate kinase (type L) of rat liver.

Synthetic peptides, representing part of the phosphorylatable site of rat liver pyruvate kinase, were phosphorylated by (³²P)ATP and the catalytic subunit of cyclic AMP-stimulated protein kinase. The shortest peptide which could be significantly phosphorylated was Arg-Arg-Ala-Ser-Val, with an apparent K_m of 0.08 mM. The apparent K_m for Arg-Arg-Ala-Ser-Val-Ala was 0.02 mM and that for Leu-Arg-Arg-Ala-Ser-Val-Ala was less than 0.01 mM. Peptides in which threonine was substituted for serine, or leucine for the one or the other arginine of the pentapeptide were not detectably phosphorylated. Substitution of phenylalanine for valine increased, and substitution of lysine or glycine for valine considerably decreased the rate of phosphorylation.     

Evidence for the location of the allosteric activation switch in the multisubunit phosphorylase kinase complex from mass spectrometric identification of chemically crosslinked peptides

Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, regulates glycogenolysis. Its activity, catalyzed by the gamma subunit, is tightly controlled by phosphorylation and activators acting through allosteric sites on its regulatory alpha, beta and delta subunits. Activation by phosphorylation is predominantly mediated by the regulatory beta subunit, which undergoes a conformational change that is structurally linked with the gamma subunit and that is characterized by the ability of a short chemical crosslinker to form beta-beta dimers. To determine potential regions of interaction of the beta and gamma subunits, we have used chemical crosslinking and two-hybrid screening. The beta and gamma subunits were crosslinked to each other in phosphorylated PhK, and crosslinked peptides from digests were identified by Fourier transform mass spectrometry, beginning with a search engine developed "in house" that generates a hypothetical list of crosslinked peptides. A conjugate between beta and gamma that was verified by MS/MS corresponded to crosslinking between K303 in the C-terminal regulatory domain of gamma (gammaCRD) and R18 in the N-terminal regulatory region of beta (beta1-31), which contains the phosphorylatable serines 11 and 26. A synthetic peptide corresponding to residues 1-22 of beta inhibited the crosslinking between beta and gamma, and was itself crosslinked to K303 of gamma. In two-hybrid screening, the beta1-31 region controlled beta subunit self-interactions, in that they were favored by truncation of this region or by mutation of the phosphorylatable serines 11 and 26, thus providing structural evidence for a phosphorylation-dependent subunit communication network in the PhK complex involving at least these two regulatory regions of the beta and gamma subunits. The sum of our results considered together with previous findings implicates the gammaCRD as being an allosteric activation switch in PhK that interacts with all three of the enzyme's regulatory subunits and is proximal to the active site cleft.     

The use of ubiquitin-peptide extensions as protein kinase substrates

Four ubiquitin-peptide extensions prepared as cloned products in E. coli were tested as casein kinase II substrates. Two extensions containing the sequence Ser-Glu-Glu-Glu-Glu-Glu were readily phosphorylated by partially purified rabbit reticulocyte casein kinase II. The other two fusion proteins, which lack a consensus phosphorylation site for casein kinase II, did not serve as substrates under identical reaction conditions. Native ubiquitin was not phosphorylated by reticulocyte casein kinase II, nor have we observed its phosphorylation in crude extracts from HeLa cells, mouse liver, or Xenopus eggs. Ubiquitin's apparent lack of phosphorylatable residues coupled with its remarkable heat stability and rapid migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels make the protein an attractive carrier for carboxyl-terminal peptides containing specific phosphorylation sites. Such ubiquitin extension proteins should prove valuable as protein kinase substrates.     

Mechanistic studies of the T4 DNA (gp41) replication helicase: functional interactions of the C-terminal Tails of the helicase subunits with the T4 (gp59) helicase loader protein

We compare the activities of the wild-type (gp41WT) and mutant (gp41delta C20) forms of the bacteriophage T4 replication helicase. In the gp41delta C20 mutant the helicase subunits have been genetically truncated to remove the 20 residue C-terminal tail peptide domains present in the wild-type enzyme. Here, we examine the interactions of these helicase forms with the T4 gp59 helicase loader and the gp32 single-stranded DNA binding proteins, both of which are physically and functionally coupled with the helicase in the T4 DNA replication complex. We show that the wild-type and mutant forms of the helicase do not differ in their ability to assemble into dimers and hexamers, nor in their interactions with gp61 (the T4 primase). However they do differ in their gp59-stimulated unwinding activities and in their abilities to translocate along a ssDNA strand that has been coated with gp32. We demonstrate that functional coupling between gp59 and gp41 involves direct interactions between the C-terminal tail peptides of the helicase subunits and the loading protein, and measure the energetics and kinetics of these interactions. This work helps to define a gp41-gp59 assembly pathway that involves an initial interaction between the C-terminal tails of the helicases and the gp59 loader proteins, followed by a conformational change of the helicase subunits that exposes new interaction surfaces, which can then be trapped by the gp59 protein. Our results suggest that the gp41-gp59 complex is then poised to bind ssDNA portions of the replication fork. We suggest that one of the important functions of gp59 may be to aid in the exposure of the ssDNA binding sites of the helicase subunits, which are otherwise masked and regulated by interactions with the helicase carboxy-terminal tail peptides.