Characterization of the minimal DNA-binding domain of the HIV integrase protein.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.     

The putative transposase of transposable element Ac from Zea mays L. interacts with subterminal sequences of Ac.

The Ac-specific ORFa protein, overexpressed in a baculovirus system, specifically binds to several subterminal fragments of Ac. The 11 bp long inverted repeats of the transposable element are not bound by the ORFa protein. Major ORFa protein-binding sites were delineated on 60 and 70 bp long sequence segments that lie 100 bp inside of the 5' Ac terminus and 40 bp inside of the 3' terminus respectively. Within all strongly bound fragments, and particularly in these 60 or 70 bp long segments, the hexamer motif AAACGG is repeated several times in direct or inverted orientation. The ORFa protein binds to synthetic concatemers of this motif, whereas the mutant motif AAAGGG is not complexed. Methylation of the cytosine residues in the AAACGG motif and/or its complementary strand has pronounced effects: whereas one of the two hemimethylated sequences has a higher affinity to the ORFa protein than both unmethylated and holomethylated DNAs, the other hemimethylated DNA is virtually not complexed at all. The native ORFa protein binding sites are more complex than the AAACGG sequence: certain Ac and Ds1 fragments devoid of AAACGG motifs (but containing several similar sequences) are weakly bound by the ORFa protein.     

Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis.

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.     

Deletion mutagenesis of the Escherichia coli UvrA protein localizes domains for DNA binding, damage recognition, and protein-protein interactions.

The UvrA protein is the DNA binding and damage recognition subunit of the damage-specific UvrABC endonuclease. In addition, it is an ATPase/GTPase, and the binding energy of ATP is linked to dimerization of the UvrA protein. Furthermore, the UvrA protein interacts with the UvrB protein to modulate its activities, both in solution and in association with DNA, where the UvrAB complex possesses a helicase activity. The domains of the UvrA protein that sponsor each of these activities were localized within the protein by studying the in vitro properties of a set of purified deletion mutants of the UvrA protein. A region located within the first 230 amino acids was found to contain the minimal region necessary for interactions with UvrB, the UvrA dimerization interface was localized to within the first 680 amino acids, and the DNA binding domain lies within the first 900 amino acids of the 940-amino acid UvrA protein. Two damage recognition domains were detected. The first domain, which coincides with the DNA binding region, is required to detect the damage. The second domain, located on or near the C-terminal 40 amino acids, stabilizes the protein-DNA complex when damage is encountered.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.     

Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA

The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.     

Domain organization of the adenovirus preterminal protein.

In adenovirus-infected cells, the virus-encoded preterminal protein and DNA polymerase form a heterodimer that is directly involved in initiation of DNA replication. Monoclonal antibodies were raised against preterminal protein, and epitopes recognized by the antibodies were identified by using synthetic peptides. Partial proteolysis of preterminal protein reveals that it has a tripartite structure, with the three domains being separated by two protease-sensitive areas, located at sites processed by adenovirus protease. These areas of protease sensitivity are probably surface-exposed loops, as they are the sites, along with the C-terminal region of preterminal protein, recognized by the monoclonal antibodies. Preterminal protein is protected from proteolytic cleavage when bound to adenovirus DNA polymerase, suggesting either multiple contact points between the proteins or a DNA polymerase-induced conformational change in preterminal protein. Two of the preterminal protein-specific antibodies induced dissociation of the preterminal protein-adenovirus DNA polymerase heterodimer and inhibited initiation of adenovirus DNA replication in vitro. Antibodies binding close to the primary processing sites of adenovirus protease inhibited DNA binding, consistent with UV cross-linking results which reveal that an N-terminal, protease-resistant domain of preterminal protein contacts DNA. Monoclonal antibodies recognizing epitopes within the C-terminal 60 amino acids of preterminal protein stimulate DNA binding, an effect mediated through a decrease in the dissociation rate constant. These results suggest that preterminal protein contains a large, noncontiguous surface required for interaction with DNA polymerase, an N-terminal DNA binding domain, and a C-terminal regulatory domain.     

Determinants of affinity and mode of DNA binding at the carboxy terminus of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1.

The role of the carboxy-terminal amino acids of the bacteriophage SPO1-encoded type II DNA-binding protein, TF1, in DNA binding was analyzed. Chain-terminating mutations truncating the normally 99-amino-acid TF1 at amino acids 96, 97, and 98 were constructed, as were missense mutations substituting cysteine, arginine, and serine for phenylalanine at amino acid 97 and tryptophan for lysine at amino acid 99. The binding of the resulting proteins to a synthetic 44-bp binding site in 5-(hydroxymethyl)uracil DNA, to binding sites in larger SPO1 [5-(hydroxymethyl)uracil-containing] DNA fragments, and to thymine-containing homologous DNA was analyzed by gel retardation and also by DNase I and hydroxy radical footprinting. We conclude that the C tail up to and including phenylalanine at amino acid 97 is essential for DNA binding and that the two C-terminal amino acids, 98 and 99, are involved in protein-protein interactions between TF1 dimers bound to DNA.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.