Binding and reversible denaturation of double-stranded DNA by Ff gene 5 protein

The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+).     

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.     

Rett syndrome-causing mutations in human MeCP2 result in diverse structural changes that impact folding and DNA interactions

Most cases of Rett syndrome (RTT) are caused by mutations in the methylated DNA-binding protein, MeCP2. Here, we have shown that frequent RTT-causing missense mutations (R106W, R133C, F155S, T158M) located in the methylated DNA-binding domain (MBD) of MeCP2 have profound and diverse effects on its structure, stability, and DNA-binding properties. Fluorescence spectroscopy, which reports on the single tryptophan in the MBD, indicated that this residue is strongly protected from the aqueous environment in the wild type but is more exposed in the R133C and F155S mutations. In the mutant proteins R133C, F155S, and T158M, the thermal stability of the domain was strongly reduced. Thermal stability of the wild-type protein was increased in the presence of unmethylated DNA and was further enhanced by DNA methylation. DNA-induced thermal stability was also seen, but to a lesser extent, in each of the mutant proteins. Circular dichroism (CD) of the MBD revealed differences in the secondary structure of the four mutants. Upon binding to methylated DNA, the wild type showed a subtle but reproducible increase in alpha-helical structure, whereas the F155S and R106W did not acquire secondary structure with DNA. Each of the mutant proteins studied is unique in terms of the properties of the MBD and the structural changes induced by DNA binding. For each mutation, we examined the extent to which the magnitude of these differences correlated with the severity of RTT patient symptoms.     

An IncY plasmid-encoded single-stranded DNA-binding protein from Escherichia coli shows the identical pattern of stacked tryptophan residues as the chromosomal ssb gene product

In an extension of earlier studies on the Escherichia coli plasmid-encoded single-stranded DNA-binding proteins pIP71a SSB, F SSB and R64 SSB [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) Biochemistry 26, 3347-3354; Casas-Finet, J. R., Khamis, M. I., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) J. Biol. Chem. 262, 8574-8593], we have investigated the binding of pIP231a SSB to natural and heavy-atom-derivatized single-stranded homopolynucleotides. Fluorimetric equilibrium binding isotherms indicate that pIP231a SSB has a greater solubility at low ionic strength than any other plasmid SSB protein investigated. Furthermore, its complex with mercurated poly(uridylic acid) [poly(Hg5U)] shows a greater resistance to disruption by salt than the other plasmid SSB complexes. Essentially complete binding of pIP231a SSB to poly(Hg5U) could be achieved, and time-resolved optically detected triplet-state magnetic resonance (ODMR) techniques could be applied to the complex. These methods allowed complete resolution of the three Trp chromophores of pIP231a SSB. Comparison of wavelength-selected ODMR results with those obtained for the poly(Hg5U) complex of a point-mutated chromosomal ssb gene product (Eco SSB) carrying substitutions of Phe for Trp [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Murphy, J. B. & Chase, J. W. (1987) J. Biol. Chem. 262, 10938-10945] confirm that Trp40 and Trp54 of pIP231a SSB are stacked in the complex, while Trp88 is not. This is the same distribution of stacked Trp residues found in Eco SSB. These results are confirmed further by specific effects observed on the ODMR signals of pIP231a SSB upon binding to poly(Br5U) and poly(dT), which are known to be caused by the stacking of Trp54 with nucleic acid bases.     

Electrostatic potential distribution of the gene V protein from Ff phage facilitates cooperative DNA binding: a model of the GVP-ssDNA complex.

The crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved for the wild-type and two mutant (Y41F and Y41H) proteins at high resolution. The Y41H mutant crystal structure revealed crystal packing interactions, which suggested a plausible scheme for constructing the polymeric protein shell of the GVP-single-stranded DNA (ssDNA) complex (Guan Y, et al., 1994, Biochemistry 33:7768-7778). The electrostatic potentials of the isolated and the cooperatively formed protein shell have been calculated using the program GRASP and they revealed a highly asymmetric pattern of the electrostatic charge distribution. The inner surface of the putative DNA-binding channel is positively charged, whereas the opposite outer surface is nearly neutral. The electrostatic calculation further demonstrated that the formation of the helical protein shell enhanced the asymmetry of the electrostatic distribution. A model of the GVP-ssDNA complex with the n = 4 DNA-binding mode could be built with only minor conformational perturbation to the GVP protein shell. The model is consistent with existing biochemical and biophysical data and provides clues to the properties of GVP, including the high cooperatively of the protein binding to ssDNA. The two antiparallel ssDNA strands form a helical ribbon with the sugar-phosphate backbones at the middle and the bases pointing away from each other. The bases are stacked and the Phe 73 residue is intercalated between two bases. The optimum binding to a tetranucleotide unit requires the participation of four GVP dimers, which may explain the cooperativity of the GVP binding to DNA.     

The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7

Gene 2.5 of bacteriophage T7 encodes a single-stranded DNA-binding protein that is essential for viral survival. Its crystal structure reveals a conserved oligosaccharide/oligonucleotide binding fold predicted to interact with single-stranded DNA. However, there is no experimental evidence to support this hypothesis. Recently, we reported a genetic screen for lethal mutations in gene 2.5 that we are using to identify functional domains of the gene 2.5 protein. This screen uncovered a number of mutations that led to amino acid substitutions in the proposed DNA binding domain. Three variant proteins, gp2.5-Y158C, gp2.5-K152E, and gp2.5-Y111C/Y158C, exhibit a decrease in binding affinity for oligonucleotides. A fourth, gp2.5-K109I, exhibits an altered mode of binding single-stranded DNA. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta26C, binds single-stranded DNA 10-fold more tightly than the wild-type protein. The three altered proteins defective in single-stranded DNA binding cannot mediate the annealing of homologous DNA, whereas gp2.5-Delta26C mediates the reaction more effectively than does wild-type. Gp2.5-K109I retains this annealing ability, albeit slightly less efficiently. With the exception of gp2.5-Delta26C, all variant proteins form dimers in solution and physically interact with T7 DNA polymerase.     

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C‐terminal domain

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Mutational analysis of amiloride sensitivity of the NhaA Na+/H+ antiporter from Vibrio parahaemolyticus.

The activity of the NhaA Na+/H+ antiporter of Vibrio parahaemolyticus is inhibited by amiloride. We found an amino acid sequence in the NhaA that was identical to a putative amiloride binding domain of the Na+/H+ exchanger in mammalian cells. We constructed mutant NhaAs that had amino acid substitutions in the putative amiloride binding domain by site-directed mutagenesis. These include V62L (Val62 replaced by Leu), F63Y, F64Y, and L65F. Most mutant NhaAs showed decreased sensitivity for amiloride. Among these, the F64Y mutant NhaA showed the least amiloride sensitivity, with a Ki value 7 to 10 times greater than that in the wild type. Thus, the sequence between residues V62 and L65 in NhaA, especially F64, is very important for the inhibitory effect of amiloride on the antiporter.     

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X-ray crystallography.

The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 A resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.     

Functional analysis of the Notch ligand Jagged1 missense mutant proteins underlying Alagille syndrome

Heterozygous mutations in the JAG1 gene, encoding Notch ligand Jagged1, cause Alagille syndrome (ALGS). As most of the mutations are nonsense or frameshift mutations producing inactive truncated proteins, haplo-insufficiency is considered the major pathogenic mechanism of ALGS. However, the molecular mechanisms by which the missense mutations cause ALGS remain unclear. Here we analyzed the functional properties of four ALGS missense mutant proteins, P163L, R184H, G386R and C714Y, using transfected mammalian cells. P163L and R184H showed Notch-binding activities similar to that of the wild-type when assessed by immunoprecipitation. However, their trans-activation and cis-inhibition activities were almost completely impaired. These mutant proteins localized mainly to the endoplasmic reticulum (ER), suggesting that the mutations induced improper protein folding. Furthermore, the mutant proteins bound more strongly to the ER chaperone proteins calnexin and calreticulin than the wild-type did. C714Y also localized to the ER, but possessed significant trans-activation activity and lacked enhanced binding to the chaperones, indicating a less severe phenotype. The properties of G386R were the same as those of the wild-type. Dominant-negative effects were not detected for any mutant protein. These results indicate that accumulation in the ER and binding to the chaperones correlate with the impaired signal-transduction activities of the missense mutant proteins, which may contribute to the pathogenic mechanism of ALGS. Our findings, which suggest the requirement for cell-surface localization of Jagged1 for cis-inhibition activities, also provide important information for understanding the molecular basis of Notch-signaling pathways.     

Six X-linked agammaglobulinemia-causing missense mutations in the Src homology 2 domain of Bruton's tyrosine kinase: phosphotyrosine-binding and circular dichroism analysis

Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequences and thereby mediate their association to ligands. Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations cause a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA). Mutations have been found in all Btk domains, including SH2. We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q. Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA. Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical. The binding of XLA mutation-containing SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% of that for the native SH2 domain. The solubility of all the mutated proteins was remarkably reduced. SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).     

Osmium tetroxide reactivity of DNA bases in nucleotide sequencing and probing of DNA structure.

Osmium tetroxide, 2,2'-bipyridine (Os,bipy) has been widely applied as a probe of the DNA structure. To obtain information about reactivity of DNA bases toward this probe synthetic homopolynucleotides poly(dT), poly(dC), poly(dG) and poly(dA) were treated with Os,bipy and the content of modified bases measured by stripping voltammetry and absorption spectrophotometry. After 20 hours' treatment strong modification of poly(dT) and poly(dC) and weak modification of poly(dG) were observed, while no modification was detected in poly(dA). At short incubation times under conditions close to those usually used in probing the DNA structure the extent of poly(dT) modification was more than 10 times higher than that of poly(dC). Thus, in single-stranded DNA Os,bipy reacts with T much greater than C and G. Due to the fast reaction of thymines with Os,bipy (and osmium tetroxide, pyridine) these chemicals can be applied in Maxam-Gilbert nucleotide sequencing as agents specific for thymines in single-stranded DNA.     

Evidence for leucine zipper motif in lactose repressor protein

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.