The linker of calmodulin lacking Glu84 is elongated in solution,although it is bent in the crystal

The solution structure of a mutant calmodulin (des84) lacking Glu84 in the central helix linking the two calmodulin lobes is substantially different from its crystal structure. As determined by small-angle X-ray scattering, the radius of gyration and the maximum linear dimension of des84 in the presence of 0.1 mM calcium are 20.8 Å and 62.5 Å, respectively. These respective dimensions are larger than those expected from the crystal structure of des84, 18.5 Å and 55.0 Å, and smaller than those expected from the crystal structure of wild type, 22.8 Å and 67.5 Å. The distance distribution function of des84 indicates that it assumes an elongated, dumbbell shape in solution. The solution scattering profile of des84 is indistinguishable from that of wild-type calmodulin. The calcium-dependent binding of melittin to des84 causes a change in its shape from elongated to spherical, as seen with other calmodulins. In comparison with calcium-saturated des84, calcium-free des84 is slightly elongated; a slight compaction is observed with native calmodulin. The observed differences between the averaged solution structure and the crystal structure of des84 suggests that an ensemble of structures is available to calmodulin in solution and that its target need not induce a change in its conformation. These results support the theory that the linker region of the central helix of calmodulin functions as a flexible tether. © 1996 Wiley-Liss, Inc.     

The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding

Using site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix. These are CaM delta 84, Glu-84 removed; CaM delta 83-84, Glu-83 and Glu-84 removed; and CaM delta 81-84, Ser-81 through Glu-84 removed. The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Similar results were obtained with all three deletion proteins. Vm values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin. Relative to bacterial control values, changes in Kact or Kd values associated with the deletions are all less than an order of magnitude: Kact values for NAD kinase and myosin light chain kinase are increased 5-7-fold, Kd values for binding of the synthetic peptide are increased 4-7-fold, and Kact values for calcineurin are increased only 1-3-fold. In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed. With NAD kinase, Kact values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and Vm values are increased by 50%; with myosin light chain kinase, Kact values are increased 2-fold and Vm values are decreased 10-15% relative to those values obtained with bovine calmodulin. These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells. No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding. Our observations indicate that contacts with the deleted residues, Ser-81 through Glu-84, are not critical in the calmodulin-target complexes we have evaluated. Formation of these calmodulin-target complexes also does not appear to be greatly affected by the global alterations in the structure of calmodulin that are associated with the deletions. In models in which the central helix is maintained in the altered calmodulins, each deleted residue causes the two lobes of calmodulin to be twisted 100 degrees relative to one another and brought 1.5 A closer together.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of the interdomain linker probed by kinetics of CO ligation to an endothelial nitric oxide synthase mutant lacking the calmodulin binding peptide (residues 503-517 in bovine)

Oxygenase and reductase domains in nitric oxide synthase are linked by a peptide region that binds calmodulin. Here we study the effects of modifying the length of the interdomain linker in a deletion mutant lacking 15 amino acids (residues 503-517) in bovine eNOS. The kinetics of CO ligation with the mutant were determined in the presence and absence of tetrahydrobiopterin and arginine and compared with the CO binding kinetics of wild-type eNOS and the eNOS oxygenase domain. In the mutant, electron flow is interrupted. The association kinetics of CO with both mutant and wild-type eNOS can be approximated with two kinetic phases, but the relative proportions change in the mutant. Both the abrogation of electron flow in the mutant and the differences in CO binding may be explained by an alteration in the docking of the FMN domain to the heme domain. We propose that the calmodulin binding residues form a helix that is critical for the proper alignment of the adjacent reductase and oxygenase domains within the active eNOS dimer in achieving proper electron transfer between them.     

Central helix role in the contraction-relaxation switching mechanisms of permeabilized skeletal and smooth muscles with genetic manipulation of calmodulin

A prominent common feature of calmodulin and troponin structures is the unusually long central helix which separates the two lobes, each containing two Ca2(+)-binding sites. To study the role of certain highly conserved residues in the helix in the contraction-relaxation switching mechanism in muscle, we measured the Ca2(+)-activated force of permeabilized skeletal and smooth muscles with three genetically manipulated forms of calmodulin. Mutated calmodulin was made to substitute for troponin-C in vertebrate skeletal fiber. The mutants had 1-4 deletions in the conserved cluster (positions 81-84) in the solvent-exposed region of the central helix, which also substantially shortened the helix. The force of the maximally activated fiber was found to be diminished only with the mutant in which the entire cluster Ser-81 to Glu-84 (CaM delta 81-84) was deleted. All such deletions were found to be completely ineffective in blocking the Ca2(+)-switching process in smooth muscle strips. The results show for the first time that at least a part of the highly conserved four-residue cluster in the central helix is critical for the contraction mechanism of striated muscle. Further, the possibility is raised that the reduced length of the central helix may be a determining factor in the Ca2(+)-switching mechanism in fast-twitch muscle. These findings combined with the results on smooth muscle indicate diversity in the structure-function specifications for the central helix of calmodulin for different target proteins.     

Mutational analysis of the linker region of EnvZ, an osmosensor in Escherichia coli.

EnvZ, a transmembrane signal transducer, is composed of a periplasmic sensor domain, transmembrane domains, and a cytoplasmic signaling domain. Between the second transmembrane domain and the cytoplasmic signaling domain there is a linker domain consisting of approximately 50 residues. In this study, we investigated the functional role of the EnvZ linker domain with respect to signal transduction. Amino acid sequence alignment of linker regions among various bacterial signal transducer proteins does not show a high sequence identity but suggests a common helix 1-loop-helix 2 structure. Among several mutations introduced in the EnvZ linker region, it was found that hydrophobic-to-charged amino acid substitutions in helix 1 and helix 2 and deletions in helix 1, loop, and helix 2 (delta14, delta8, and delta7) resulted in constitutive OmpC expression. In the linker mutant EnvZ x delta7, both kinase and phosphatase activities were significantly reduced but the ratio of kinase to phosphatase activity increased, consistent with the constitutive OmpC expression. In contrast, the purified cytoplasmic fragment of EnvZ x delta7 possessed both kinase and phosphatase activities at levels similar to those of the cytoplasmic fragment of wild-type EnvZ. In addition, the linker mutations had no direct effect on EnvZ C-terminal dimerization. These results together with previous data suggest that the linker region is not directly involved in EnvZ enzymatic activities and that it may have a crucial role in propagating a conformational change to ensure correct positioning of two EnvZ molecules within a dimer during the transmembrane signaling.     

Yeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate.

Using small-angle X-ray scattering from solutions of yeast hexokinase, we have measured the radii of gyration of the monomeric B isozyme and its complexes with sugar substrates. We find that the radius of gyration decreases by 0.95 +/- 0.24 A upon binding glucose and 1.25 +/- 0.28 A upon binding glucose 6-phosphate. This observed reduction in radius of gyration in the presence of glucose is the same as that calculated from the coordinates of the high-resolution crystal structures of native hexokinase B and a glucose complex with hexokinase A. Thus, these measurements suggest that the dramatic closing of the slit between the two lobes of hexokinase observed in the crystal structures (Bennett, W.S., & Steitz, T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848--4852) occurs in solution when either glucose or glucose 6-phosphate is bound.     

High-affinity calmodulin binding is required for the rapid entry of Bordetella pertussis adenylyl cyclase into neuroblastoma cells.

Bordetella pertussis produces a calmodulin-stimulated adenylyl cyclase that invades animal cells and raises intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24, 6323-6328]. The mechanism for invasion of animal cells by this enzyme has not been defined, but there is considerable evidence that it does not enter by receptor-mediated endocytosis [Gordon, V. M., Leppla, S. H., & Hewlett, E. L. (1988) Infect. Immun. 56, 1066-1069; Donovan, M. G., & Storm, D. R. (1990) J. Cell. Physiol. 145, 444-449]. In this study, the importance of high-affinity calmodulin (CaM) binding for entry of the enzyme into neuroblastoma cells was evaluated using a mutant enzyme that has significantly lower affinity for calmodulin than the wild-type enzyme. Oligonucleotide-directed site-specific mutagenesis was used to create a point mutant at a critical tryptophan residue (Trp-242) within the proposed CaM binding domain of the B. pertussis adenylyl cyclase. Substitution of Trp-242 with Glu lowered the apparent affinity of the enzyme for calmodulin by 250-fold; however, the maximal enzyme activity in the presence of saturating calmodulin was equivalent to the wild-type enzyme. The Glu-242 mutant adenylyl cyclase was returned to B. pertussis by homologous recombination, and the enzyme produced by this strain was examined for invasion of neuroblastoma cells. Although the mutant enzyme stimulated the production of intracellular cAMP in neuroblastoma cells, the rate of cAMP accumulation was at least 10-fold lower than that caused by the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new assay for functional screening of BRCA2 linker region mutations identifies variants that alter chemoresistance to cisplatin

Variants of unknown significance (VUS) complicate the assignment of risk to new DNA sequence variants found in at-risk populations. This study focused on the poorly studied linker region of the cancer-associated BRCA2 protein encoded by exons twelve through fourteen of BRCA2. To develop a new method to characterize VUS in this region of BRCA2, we first chose to study 4 reported VUS occurring on evolutionarily conserved residues within the linker region. To determine if these VUS represent neutral changes or if they impact the function of the BRCA2 protein, we stably transfected expression plasmids encoding wild-type or each mutant peptide into T47D breast cancer cells, which are wild-type for BRCA2. Four mutant peptide expressing cell lines and a wild-type linker region expressing cell line next were studied by challenging transfected cell lines with the DNA crosslinking compound cisplatin (10 μM) for 5 days. Expression of the wild-type linker region and certain mutant linker peptides (N2452D and I2285V) decreased apoptosis (as demonstrated by cell death detection assay) in transfected cell lines, indicating that the linker region peptide directly or indirectly affects the DNA damage repair pathway. By determining the cell survival and assaying the apoptotic index of treated cell lines, one could potentially use this screen to determine that a particular VUS has a functional impact on BRCA2 function, and hence is of functional significance. We conclude that this method is useful for screening the effect of linker region VUS on BRCA2 function, and to identify mutations for further testing. We also conclude that mutations in the linker region may have heretofore unappreciated roles in BRCA2 function.     

Specific conformation and Ca(2+)-binding mode of yeast calmodulin: insight into evolutionary development

The vertebrate calmodulin is configured with two structurally independent globular lobes in N- and C-terminus, and a flexible central linker. Distinctly, two lobes of calmodulin from Saccharomyces cerevisiae (yCaM) interact and influence the Ca(2+)-binding profile of each other. We explored this further using the mutant proteins with eliminated Ca(2+)-binding ability in one of the lobes and found that the Ca(2+)-bound N-lobe associates with the Ca(2+)-free C-lobe to gain the Ca(2+) affinity of a wild-type level. Next, analysing series of C-terminal residue truncation mutant, we found that the truncation of C-terminal three residues induce the hyper Ca(2+) affinity. These residues are also important for the general structural behaviour of calmodulin, such as Ca(2+)-induced slow mobility shift in polyacrylamide gel electrophoresis and for the ability to activate Cmk1p (yeast calmodulin kinase). These suggest: (i) when Ca(2+) occupies only N-lobe, two lobes interact and form the stable intermediate leading to a proper level of Ca(2+) affinity; (ii) the C-terminal three residues are required to prohibit abnormal stabilization of the intermediate promoting abnormally high Ca(2+) affinity and for recognition of target enzymes. A model for Ca(2+) and target bindings of yCaM is proposed. Evolutional aspect concerning the biological significance of this model was discussed.     

Calcium-induced shape change of calmodulin with mastoparan studied by solution X-ray scattering

Solution x-ray scattering using synchrotron radiation as an x-ray source was used to analyze the Ca2+-dependent shape change of pig brain calmodulin in detail. The radius of gyration of calmodulin at 10 mg/ml was increased by 0.9 A. The increase was nearly completed when 2.5 mol of Ca2+/mol of calmodulin was added, whereas the radius of gyration of calmodulin with mastoparan decreased by about 3 A with an increasing Ca2+ concentration up to 4 mol of Ca2+/mol of calmodulin. At a moderate angle of region, both scattering profiles from calmodulin with or without Ca2+ displayed clear humps at s = 0.03 A-1 which are characteristic of a dumbbell structure. However, in the presence of mastoparan, the hump in the scattering profile became obscure and later disappeared with the third and fourth Ca2+ binding to calmodulin. These findings are attributable to the Ca2+-induced shape change of calmodulin with mastoparan from a dumbbell structure to a non-dumbbell structure in which the distance between the two lobes of calmodulin become closer by a bend in the central helix.     

Role of the linker region of the anion-stimulated ATPase ArsA. Effect of deletion and point mutations in the linker region

The anion-stimulated ATPase ArsA in Escherichia coli consists of two homologous halves, A1 and A2, which are connected by a 40-amino acid long stretch of residues designated as the linker region. The linker region of ArsA lies in close proximity of the nucleotide-binding domain(s) of ArsA and is involved in significant conformational changes on binding of the substrates. Hence, it has been suggested earlier that the linker may play an important role in the function of ArsA. The aim of the present study was to determine the role of the linker by deletion and by site-directed mutagenesis of specific residues. Effect of deletion of the linker was determined by using the in vivo complementation approach where two halves of ArsA were co-expressed either with or without the linker region. Two co-expressed halves of ArsA conferred arsenite resistance only if the linker region was present on one of the halves. Of the six different point mutations created in the linker region, three (G284S, R290S, and D303G) were seen to drastically affect the catalytic function of ArsA. In addition, these three mutant alleles conferred arsenite sensitivity in cells carrying the wild type arsB gene. Trypsin proteolysis studies carried out with the purified proteins showed that the A1 nucleotide-binding domain in D303G protein has a conformation different from the wild type ArsA, suggesting that the linker region interacts with the nucleotide-binding domain(s) of ArsA. Based on the studies presented here, we propose that, in addition to providing flexibility, the nature of the residues themselves in the linker region is important for the conformation of the nucleotide-binding domains and for the catalytic function of ArsA.     

Modulation of the Shaker K(+) channel gating kinetics by the S3-S4 linker

In Shaker K(+) channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3-S4 linker. To investigate the role of the S3-S4 linker in the ShakerH4Delta(6-46) (ShakerDelta) K(+) channel activation, we constructed S3-S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K(+) currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerDelta. The apparent number of gating charges was 12.6+/-0.6 e(o) in ShakerDelta, 12.7+/-0.5 in 10 aa linker, and 12.3+/-0.9 in 5 aa linker channels, but it was only 5.6+/-0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P(o)(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the ShakerDelta K(+) channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3-S4 linker plays an important role in setting ShakerDelta channel activation and deactivation kinetics.     

Association of melittin with the isolated myosin light chains

Melittin is a 26-residue peptide which undergoes high-affinity calcium-dependent binding by calmodulin [Barnette, M.S., Daly, R., & Weiss, B. (1983) Biochem. Pharmacol. 32, 2929; Comte, M., Maulet, Y., & Cox, J.A. (1983) Biochem. J. 209, 269; Anderson, S.R., & Malencik, D.A. (1986) Calcium Cell Funct. 6, 1]. The results in this paper show that three different types of myosin light chain--the smooth muscle regulatory light chain, the smooth muscle essential light chain, and the skeletal muscle regulatory 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) light chain--also associate with melittin. The resulting complexes have dissociation constants ranging from 1.1 to 2.5 microM in the presence of 0.10 M NaCl and from approximately 50 to approximately 130 nM in solutions of 20 mM 3-(N-morpholino)propanesulfonic acid alone. The regulatory smooth muscle myosin light chain exhibits two equivalent melittin binding sites while each of the others displays only one. The myosin light chains evidently contain elements of structure related to the macromolecular interaction sites present in calmodulin and troponin C but not in parvalbumin. The association of melittin and other peptides with the light chains requires consideration whenever assays of the calmodulin-dependent activity of myosin light chain kinase are used to determine peptide binding by calmodulin. The binding measurements performed on the DTNB light chain and melittin necessitated derivation of the equation relating complex formation to the observed fluorescence anisotropy of a solution containing three fluorescent components. This analysis is generally applicable to equilibria involving the association of two fluorescent molecules emitting in the same wavelength range.     

Kinetic and magnetic resonance studies of the glutamate-43 to serine mutant of staphylococcal nuclease

The Glu-43 residue of staphylococcal nuclease has been proposed to function as a general base that facilitates the attack of water on the phosphodiester substrate [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555]. With DNA as substrate, Vmax in the glutamate-43--serine (E43S) mutant enzyme is decreased by 2700-fold at pH 7.4 but only 376-fold at pH 9.9. With the wild-type enzyme, Vmax increases with pH to pH 9.2, above which it becomes less sensitive to further increase in pH, leveling off at pH 9.8. In contrast, Vmax of the E43S mutant continues to rise, first order in [OH-], to pH 9.8. Above pH 10 both activities fall irreversible. Hence the hydroxyl ion can partially replace the effect of Glu-43 on kcat, in accord with the proposed role of Glu-43 as a general base. The inflection point in the curve relating pH to log Vmax of the wild-type enzyme at pH 9.4 may reflect the ionization of a Ca2+-bound water, or of a Lys or Tyr residue at the active site. The activator Ca2+ and the competitive inhibitor Mn2+ bind to the E43S mutant an order of magnitude more weakly than to the wild-type enzyme as detected by kinetics and by direct metal binding studies, and approximately one additional water ligand on Mn2+ is found in the binary Mn2+ complex of the E43S mutant (1.4 +/- 0.2) as compared to that of the wild-type enzyme (0.8 +/- 0.2). These data suggest that Glu-43 coordinates the divalent cation in the binary enzyme-metal complex but dissociates from the metal to create a water binding site and to function as a general base in the ternary enzyme-metal-DNA complex. While a 2-fold weaker binding of DNA to the Ca2+ complex of the E43S mutant than to the wild-type enzyme is found by kinetic studies, an order of magnitude tighter binding of the competitive inhibitor 3',5'-pdTp to the Mn2+ and Ca2+ complexes of E43S is found by direct binding studies. Distances from Co2+ to phosphorus in the ternary enzyme-Co2+-pdTp complexes reveal coordination of only the 5'-phosphate by Co2+ on the wild-type enzyme but coordination of both the 3'- and 5'-phosphates of pdTp on the E43S mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Point mutation of a tyrosine in the linker region of Syk results in a gain of function

The protein tyrosine kinase Syk plays an essential role in Fc epsilon RI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the Fc epsilon RI-mediated tyrosine phosphorylation of phospholipase C-gamma and a dramatic enhancement of histamine release. The in vivo Fc epsilon RI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The Fc epsilon RI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.     

The interaction of calmodulin with melittin

Studies utilizing the interaction of melittin with the 1-106 fragment of calmodulin, the protection of calmodulin from tryptic digestion by melittin, and the interaction of the carbocyanine dye Stains-all with the calmodulin-melittin complex have indicated that complex formation of calmodulin with melittin involves the alpha-helical connecting bridge joining the N- and C-terminal lobes of calmodulin.     

Site-directed deletion mutants of a carboxyl-terminal region of human dihydrofolate reductase.

Substrate and inhibitor binding to dihydrofolate reductase (DHFR) primarily involves residues in the amino-terminal half of the enzyme; however, antibody binding studies performed in this laboratory suggested that the loop region located in the carboxyl terminus of human DHFR (hDHFR; residues 140-186) is involved in conformational changes that occur upon ligand binding and affect enzyme function (Ratnam, M., Tan, X., Prendergast, N.J., Smith, P.L. & Freisheim, J.H. (1988) Biochemistry 27, 4800-4804). To investigate this observation further, site-directed mutagenesis was used to construct deletion mutants of hDHFR missing 1 (del-1), 2 (del-2), 4 (del-4), and 6 (del-6) residues from loops in the carboxyl terminus of the enzyme. The del-1 mutant enzyme has a two-amino acid substitution in addition to the one-amino acid deletion. Deletion of only one amino acid resulted in a 35% decrease in the specific activity of the enzyme. The del-6 mutant enzyme was inactive. Surprisingly, the del-4 mutant enzyme retained a specific activity almost 33% that of the wild type. The specific activity of the del-2 mutant enzyme was slightly higher (38% wild-type activity) than that of the del-4 mutant. All three active deletion mutants were much less stable than the wild-type enzyme, and all three showed at least a 10-fold increase in Km values for both substrates. The del-1 and del-2 mutants exhibited a similar increase in KD values for both substrate and cofactor. The three active deletion mutants lost activity at concentrations of activating agents such as KCl, urea, and p-hydroxymercuribenzoate that continued to stimulate the wild-type enzyme. Antibody binding studies revealed conformational differences between the wild-type and mutant enzymes both in the absence and presence of bound folate. Thus, although the loops near the carboxyl terminus are far removed from the active site, small deletions of this region significantly affect DHFR function, indicating that the loop structure in mammalian DHFR plays an important functional role in its conformation and catalysis.     

Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators.     

Defect in general priming conferred by linker region mutants of Escherichia coli dnaB.

The dnaB gene of Escherichia coli encodes a bifunctional primase accessory protein/helicase necessary for chromosomal replication. Monomers of DnaB comprise two trypsin-resistant domains connected by a 45-amino-acid linker. To investigate the role of the linker in the structure and function of DnaB, we have purified and characterized three DnaB mutant proteins having single amino acid substitutions in the linker. We find that the mutant proteins retain the two-domain structure and assemble into hexamers that may be less stable than hexamers formed by wild-type DnaB. These mutant hexamers have hydrodynamic properties slightly different from those of the wild type, suggestive of a more open structure. The mutant proteins had reduced or absent ability to stimulate primase and also exhibited slight alterations in ATPase activity compared with the wild type. We conclude that the linker region promotes primase-DnaB interaction, but this effect may be indirect. We propose a model involving repositioning of N-terminal domains to explain the properties of the mutant proteins.     

Mutations in the linker domain of NBD2 of SUR inhibit transduction but not nucleotide binding

ATP-sensitive potassium (K(ATP)) channels are composed of an ATP-binding cassette (ABC) protein (SUR1, SUR2A or SUR2B) and an inwardly rectifying K(+) channel (Kir6.1 or Kir6.2). Like other ABC proteins, the nucleotide binding domains (NBDs) of SUR contain a highly conserved "signature sequence" (the linker, LSGGQ) whose function is unclear. Mutation of the conserved serine to arginine in the linker of NBD1 (S1R) or NBD2 (S2R) did not alter the ability of ATP or ADP (100 microM) to displace 8-azido-[(32)P]ATP binding to SUR1, or abolish ATP hydrolysis at NBD2. We co-expressed Kir6.2 with wild-type or mutant SUR in Xenopus oocytes and recorded the resulting currents in inside-out macropatches. The S1R mutation in SUR1, SUR2A or SUR2B reduced K(ATP) current activation by 100 microM MgADP, whereas the S2R mutation in SUR1 or SUR2B (but not SUR2A) abolished MgADP activation completely. The linker mutations also reduced (S1R) or abolished (S2R) MgATP-dependent activation of Kir6.2-R50G co-expressed with SUR1 or SUR2B. These results suggest that the linker serines are not required for nucleotide binding but may be involved in transducing nucleotide binding into channel activation.