A 22-amino-acid peptide restores DNA-binding activity to dimerization-defective mutants of the estrogen receptor.

We have identified residues within the estrogen receptor that are required for dimerization and high-affinity DNA binding. A 22-amino-acid peptide encompassing these residues was sufficient to restore DNA-binding activity to a mutant receptor lacking most of the hormone-binding domain. Point mutagenesis of the fusion protein confirmed that this sequence continued to mediate dimerization in a manner similar to that within the native receptor, although its position relative to the DNA-binding domain was appreciably altered.     

Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

KpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5'-GGTACC-3' and transfers the methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation and in vitro restriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.     

Mutational analysis of the att DNA-binding domain of phage Mu transposase.

The transposase (A protein) of phage Mu encodes binding to two families of DNA sites, att sites located at the Mu ends and enhancer sites located internally. Separate subdomains in the N-terminal domain I of Mu A protein are known to be involved in recognition of the att and enhancer sites. We have delineated an approximately 135 aa region within domain I beta gamma that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain I beta gamma as well as the intact Mu A protein. Extensive mutagenesis of residues around a putative helix-turn-helix DNA-binding motif within the I beta domain identified several mutants defective in DNA transposition in vivo. Of these, Mu A(K157Q) was completely defective in att DNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity for att DNA and low levels of transposition in vitro. Our results indicate that residues in the gamma region are required for activity and that residues outside the beta gamma region must also influence discrimination between the multiple att sites.     

Genetic analysis reveals essential and non-essential amino acids within the telomeric DNA-binding interface of Cdc13p

Cdc13p is a specific single-stranded telomeric DNA-binding protein of Saccharomyces cerevisiae. It is involved in protecting telomeres and regulating telomere length. The telomere-binding domain of Cdc13p is located between residues 497 and 693, and its structure has been resolved by NMR spectroscopy. A series of aromatic, hydrophobic and basic residues located at the DNA-binding surface of Cdc13p are involved in binding to telomeres. Here we applied a genetic approach to analyse the involvements of these residues in telomere binding. A series of mutants within the telomere-binding domain of Cdc13p were identified that failed to complement cdc13 mutants in vivo. Among the amino acids that were isolated, the Tyr522, Arg635, and Ile633 residues were shown to locate at the DNA-binding surface. We further demonstrated that Y522C and R635A mutants failed to bind telomeric DNA in vitro, indicating that these residues are indeed required for telomere binding. We did not, however, isolate other mutant residues located at the DNA-binding surface of Cdc13p beyond these three residues. Instead, a mutant on Lys568 was isolated that did not affect the essential function of Cdc13p. The Lys568 is also located on the DNA-binding surface of Cdc13p. Thus these results suggested that other DNA-binding residues are not essential for telomere binding. In the present study, we have established a genetic test that enabled the identification of telomere-binding residues of Cdc13p in vivo. This type of analysis provides information on those residues that indeed contribute to telomere binding in vivo.     

The structure of the C-terminal actin-binding domain of talin

Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.     

Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis

Phage T4 endonuclease II (EndoII), a GIY-YIG endonuclease lacking a carboxy-terminal DNA-binding domain, was subjected to site-directed mutagenesis to investigate roles of individual amino acids in substrate recognition, binding, and catalysis. The structure of EndoII was modeled on that of UvrC. We found catalytic roles for residues in the putative catalytic surface (G49, R57, E118, and N130) similar to those described for I-TevI and UvrC; in addition, these residues were found to be important for substrate recognition and binding. The conserved glycine (G49) and arginine (R57) were essential for normal sequence recognition. Our results are in agreement with a role for these residues in forming the DNA-binding surface and exposing the substrate scissile bond at the active site. The conserved asparagine (N130) and an adjacent proline (P127) likely contribute to positioning the catalytic domain correctly. Enzymes in the EndoII subfamily of GIY-YIG endonucleases share a strongly conserved middle region (MR, residues 72 to 93, likely helical and possibly substituting for heterologous helices in I-TevI and UvrC) and a less strongly conserved N-terminal region (residues 12 to 24). Most of the conserved residues in these two regions appeared to contribute to binding strength without affecting the mode of substrate binding at the catalytic surface. EndoII K76, part of a conserved NUMOD3 DNA-binding motif of homing endonucleases found to overlap the MR, affected both sequence recognition and catalysis, suggesting a more direct involvement in positioning the substrate. Our data thus suggest roles for the MR and residues conserved in GIY-YIG enzymes in recognizing and binding the substrate.     

Recognition of DNA by p53 core domain and location of intermolecular contacts of cooperative binding

We present an analysis by NMR of a 58 kDa complex of the core domain of the tumour suppressor p53 with DNA that complements and extends the crystal structure analysis. Binding of specific DNA caused significant chemical shifts of residues on the DNA-binding interface that translated into the beta-sheet of the protein. Binding of non-specific DNA caused weak but qualitatively the same shifts, corresponding to weaker binding interactions. The observed chemical shift differences correlate with frequency of cancer-inducing mutations, suggesting that the affected residues contribute to the stability of p53 core domain-DNA complex. We also identified two affected regions on the surface of the protein: helix 1 (residues V173-C182) plus G244 and residues L114-T118, which may represent a dimerisation interface.     

The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics

ErbB-2 is a member of the family of epidermal growth factor receptors, which shows an oncogenic mutation in the rat gene neu, Val664Glu in the transmembrane domain that causes permanent dimerisation and subsequently leads to uncontrollable cell division and tumour formation. We have obtained the alpha-helical structure of the mutant transmembrane domain dimer experimentally with site-specific infrared dichroism (SSID) based on six transmembrane peptides with 13C18O carbonyl group-labelled residues. The derived orientational data indicate a local helix tilt ranging from 28(+/-6) degrees to 22(+/-4) degrees. Altogether using orientational constraints from SSID and experimental alpha-helical constraints while performing a systematic conformational search including molecular dynamics simulation in a lipid bilayer, we have obtained a unique experimentally defined atomic structure. The resulting structure consists of a right handed alpha-helical bundle with the residues Ile659, Val663, Leu667, Ile671, Val674 and Leu679 in the dimerisation interface. The right-handed bundle is in contrast to the left-handed structures obtained in previous modelling efforts. In order to facilitate tight helical packing, the spacious Glu664 residues do not interact directly but with water molecules that enter the bilayer.     

Modulation of PAX6 homeodomain function by the paired domain

PAX6 is required for proper development of the eye, central nervous system, and nose. PAX6 has two DNA binding domains, a glycine-rich region that links the two DNA binding domains, and a transactivation domain. There is evidence that the different DNA binding domains of PAX6 have different target genes. However, it is not clear if the two DNA binding domains function independently. We have studied the effect of structural changes in the paired domain on the function of PAX6 mediated through its homeodomain. The R26G and I87R mutations have been reported in different human patients with clinically different phenotypes and are in the N- and the C-terminal halves of the paired domain, respectively. Surprisingly, we found that the I87R mutant protein not only lost the transactivation function but also failed to bind DNA by either of its DNA binding domains. In contrast, the R26G mutant protein lost DNA binding through its paired domain but had greater DNA binding and transactivation than wild-type PAX6 on homeodomain binding sites. Like R26G, the 5a isoform showed higher DNA binding than wild-type PAX6. This study demonstrates that the two subdomains of the paired domain influence the function of the homeodomain differentially and also provides an explanation for the difference in phenotypes associated with these mutations.     

Structural characterization of two alternate conformations in a calbindin D₉k-based molecular switch

We have demonstrated that calbindin D(9k) can be converted into a calcium-sensing switch (calbindin-AFF) by duplicating the C-terminal half of the protein (residues 44-75) and appending it to the N-terminus (creating residues 44'-75'). This re-engineering results in a ligand-driven interconversion between two native folds: the wild-type structure (N) and a circularly permuted form (N'). The switch between N and N' is predicted to involve exchange of the 44-75 and 44'-75' segments, possibly linked to their respective folding and unfolding. Here we present direct structural evidence supporting the existence of N and N'. To isolate the N' and N conformations, we introduced the knockdown Ca(2+) binding mutation Glu → Gln at position 65 (E65Q mutant) or at the analogous position 65' (E65'Q mutant). E65Q and E65'Q are therefore expected to adopt conformations N' and N, respectively, in the presence of calcium. Though the amino acid sequences of E65Q and E65'Q differ at only these two positions, nuclear magnetic resonance resonance assignments, chemical shifts, and paramagnetic relaxation enhancement data reveal that they take on separate structures when bound to calcium. Both proteins are comprised of a well-folded domain and a disordered region. However, the segment that is disordered in E65Q (residues 44-75) is folded in E65'Q, and the region that is disordered in E65'Q (residues 44'-75') is structured in E65Q. The results demonstrate that the N' N' conformational change is mediated by a mutually exclusive folding reaction in which folding of one segment of the protein is coupled to unfolding of another segment, and vice versa.     

Mapping of a DNA binding region of the PI-sceI homing endonuclease by affinity cleavage and alanine-scanning mutagenesis.

The PI-SceI protein is a member of the LAGLIDADG family of homing endonucleases that is generated by a protein splicing reaction. PI-SceI has a bipartite domain structure, and the protein splicing and endonucleolytic reactions are catalyzed by residues in domains I and II, respectively. Structural and mutational evidence indicates that both domains mediate DNA binding. Treatment of the protein with trypsin breaks a peptide bond within a disordered region of the endonuclease domain situated between residues Val-270 and Leu-280 and interferes with the ability of this domain to bind DNA. To identify specific residues in this region that are involved in DNA binding and/or catalysis, alanine-scanning mutagenesis was used to create a set of PI-SceI mutant proteins that were assayed for activity. One of these mutants, N281A, was >300-fold less active than wild-type PI-SceI, and two other proteins, R277A and N284A, were completely inactive. These decreases in cleavage activity parallel similar decreases in substrate binding by the endonuclease domains of these mutant proteins. We mapped the approximate position of the disordered region to one of the ends of the 31 base pair PI-SceI recognition sequence using mutant proteins that were substituted with cysteine at residues Asn-274 and Glu-283 and tethered to the chemical nuclease FeBABE. These mutational and affinity cleavage data strongly support a model of PI-SceI docked to its DNA substrate that suggests that one or more residues identified here are responsible for contacting base pair A/T(-)(9), which is essential for substrate binding.