Biochemical and mutational analysis of EcoRII functional domains reveals evolutionary links between restriction enzymes

The archetypal Type IIE restriction endonuclease EcoRII is a dimer that has a modular structure. DNA binding studies indicate that the isolated C-terminal domain dimer has an interface that binds a single cognate DNA molecule whereas the N-terminal domain is a monomer that also binds a single copy of cognate DNA. Hence, the full-length EcoRII contains three putative DNA binding interfaces: one at the C-terminal domain dimer and two at each of the N-terminal domains. Mutational analysis indicates that the C-terminal domain shares conserved active site architecture and DNA binding elements with the tetrameric restriction enzyme NgoMIV. Data provided here suggest possible evolutionary relationships between different subfamilies of restriction enzymes.     

Spectral properties and DNA targeting features of a thiazole orange-peptide bioconjugate

The molecular recognition features of a DNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency are described. The bioconjugate combines a polypeptide from the Tc3 transposase DNA-binding domain with the dsDNA-sensitive fluorophore thiazole orange. Fluorescence spectroscopy and circular dichroism reveal that the polypeptide moiety determines the DNA sequence specificity as the intercalating dye makes nonspecific contributions to binding affinity. The conjugated thiazole orange is able to intercalate and fluoresce when the peptide binds at concentrations where little fluorescence is observed from either the bioconjugate alone or the bioconjugate mixed with DNA lacking the target sequence. Fluorescence studies indicate this molecular probe is sequence specific, binds the native Tc3 DNA target sequence with nanomolar affinity (KD approximately 15 nM), and is able to discriminate multiple point mutations in the cognate DNA site. The attachment of a sequence-specific binding peptide onto a functional probe provides a viable strategy for construction of synthetic enzymes and repressors, and facilitates dynamic studies of protein-DNA interactions.     

Sequence recognition in the minor groove of DNA by covalently linked formamido imidazole-pyrrole-imidazole polyamides: effect of H-pin linkage and linker length on selectivity and affinity

The polyamide N-formamido imidazole-pyrrole-imidazole (f-ImPyIm) binds with an exceptionally high affinity for its cognate site 5'-ACGCGT-3' as a stacked, staggered, and noncovalent cooperative dimer. Investigations are presented into its sequence specificity and binding affinity when linked covalently as an H-pin "dimer". Five f-ImPyIm cross-linked analogues with six to nine methylene linkers and an eight-linked ethylene glycol linker were examined to investigate the effect of linkage and linker length on DNA binding. Thermal denaturation studies on short DNA hairpins showed preferential binding by both f-ImPyIm (DeltaTm = 7.8 degrees C) and its cross-linked derivatives (DeltaTm > 30 degrees C) at 5'-ACGCGT-3', indicating sequence specificity was retained on linkage. DNase I footprinting confirmed strict cognate site selectivity and demonstrated that affinity increased with linker length (f-ImPyIm-9 = f-ImPyIm-8 = f-ImPyIm-EG-8 > f-ImPyIm-7 > f-ImPyIm-6). The eight- and nine-linked derivatives bound at 100-fold lower concentrations at the cognate site relative to f-ImPyIm-6, and with 10-fold higher affinity than unlinked f-ImPyIm. Use of an ethylene glycol linkage in f-ImPyIm-EG-8 to improve solubility slightly increased the cognate site affinity relative to those of f-ImPyIm-8 and f-ImPyIm-9, although some selectivity was lost at high ligand concentration. CD demonstrated that cognate site binding by eight and nine-linked compounds occurred in the minor groove. SPR analysis gave a binding affinity (K) for f-ImPyIm-EG-8 at the cognate site of 2 x 10(10) M-1, representing a 100-fold increase relative to that of f-ImPyIm. This study demonstrates that the high-affinity cooperative binding of f-ImPyIm can be enhanced significantly by suitable covalent linkage, while maintaining its strict cognate site selectivity.     

The DNA-binding domain of human papillomavirus type 18 E1. Crystal structure, dimerization, and DNA binding

High risk types of human papillomavirus, such as type 18 (HPV-18), cause cervical carcinoma, one of the most frequent causes of cancer death in women worldwide. DNA replication is one of the central processes in viral maintenance, and the machinery involved is an excellent target for the design of antiviral therapy. The papillomaviral DNA replication initiation protein E1 has origin recognition and ATP-dependent DNA melting and helicase activities, and it consists of a DNA-binding domain and an ATPase/helicase domain. While monomeric in solution, E1 binds DNA as a dimer. Dimerization occurs via an interaction of hydrophobic residues on a single alpha-helix of each monomer. Here we present the crystal structure of the monomeric HPV-18 E1 DNA-binding domain refined to 1.8-A resolution. The structure reveals that the dimerization helix is significantly different from that of bovine papillomavirus type 1 (BPV-1). However, we demonstrate that the analogous residues required for E1 dimerization in BPV-1 and the low risk HPV-11 are also required for HPV-18 E1. We also present evidence that the HPV-18 E1 DNA-binding domain does not share the same nucleotide and amino acid requirements for specific DNA recognition as BPV-1 and HPV-11 E1.     

Increased stability and DNA site discrimination of "single chain" variants of the dimeric beta-barrel DNA binding domain of the human papillomavirus E2 transcriptional regulator

Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric beta-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.     

The GCN4 bZIP targets noncognate gene regulatory sequences: quantitative investigation of binding at full and half sites

We previously reported that a basic region/leucine zipper (bZIP) protein, a hybrid of the GCN4 basic region and C/EBP leucine zipper, not only recognizes cognate target sites AP-1 (5'-TGACTCA-3') and cAMP-response element (CRE) (5'-TGACGTCA-3') but also binds selectively to noncognate DNA sites: C/EBP (CCAAT/enhancer binding protein, 5'-TTGCGCAA), XRE1 (xenobiotic response element, 5'-TTGCGTGA), HRE (HIF response element, 5'-GCACGTAG), and E-box (5'-CACGTG). In this work, we used electrophoretic mobility shift assay (EMSA) and circular dichroism (CD) for more extensive characterization of the binding of wt bZIP dimer to noncognate sites as well as full- and half-site derivatives, and we examined changes in flanking sequences. Quantitative EMSA titrations were used to measure dissociation constants of this hybrid, wt bZIP, to DNA duplexes: Full-site binding affinities gradually decrease from cognate sites AP-1 and CRE with Kd values of 13 and 12 nM, respectively, to noncognate sites with Kd values of 120 nM to low microM. DNA-binding selectivity at half sites is maintained; however, half-site binding affinities sharply decrease from the cognate half site (Kd = 84 nM) to noncognate half sites (all Kd values > 2 microM). CD shows that comparable levels of alpha-helical structure are induced in wt bZIP upon binding to cognate AP-1 or noncognate sites. Thus, noncognate sites may contribute to preorganization of stable protein structure before binding target DNA sites. This work demonstrates that the bZIP scaffold may be a powerful tool in the design of small, alpha-helical proteins with desired DNA recognition properties.     

Autonomous folding and coenzyme binding of the excised pyridoxal 5'-phosphate binding domain of aspartate aminotransferase from Escherichia coli

The coenzyme (PLP) binding domain (residues 47-329) of the dimeric aspartate aminotransferase from Escherichia coli was produced separately by recombinant DNA methods. It folded autonomously both in vivo and in vitro, that is, independently of the native N- and C-terminal extensions that combine to form the small domain of eAAT. The PLP-domain had one binding site for PLP of relatively high affinity involving a covalent bond to the protein. It was monomeric, although the major subunit-subunit interface at the 2-fold symmetry axis remained unchanged. This effect appears to be due mainly to the absence of the N-terminal extension that contains hydrophobic residues, which interact with the PLP-domain of the second subunit in the wild-type dimer. Judged by circular dichroism, fluorescence, and HPLC gel filtration at increasing concentrations of guanidinium chloride, the PLP-domain underwent a three-state unfolding transition (M' in equilibrium M'* in equilibrium U') involving a compact intermediate M'*. This behavior parallels the unfolding of the dissociated native monomer of cAAT.     

cAMP-dependent protein kinase regulatory subunit type IIbeta: active site mutations define an isoform-specific network for allosteric signaling by cAMP

cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RIIbeta and two mutant RIIbeta subunits, engineered by converting a conserved Arg to Lys in each cAMP-binding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The Ka for RIIbeta is 610 nM, which is 10-fold greater than its Kd(cAMP) and significantly higher than for RIalpha and RIIalpha. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The Ka of the A domain mutant protein is 21-fold greater than that of wild-type and the Kd(cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant Ka is 2-fold less than its Kd(cAMP), demonstrating that, unlike RIalpha, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a Ka identical to the Kd(cAMP) of full-length RIIbeta, indicating that the B domain inhibits holoenzyme activation for RIIbeta. In RIalpha, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.     

C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2

BackgroundHuman Nucleic Acid Binding Protein 1 and 2 (hNABP1 and 2; also known as hSSB2 and 1, respectively) are two newly identified single-stranded (ss) DNA binding proteins (SSB). Both NABP1 and NABP2 have a conserved oligonucleotide/oligosaccharide-binding (OB)-fold domain and a divergent carboxy-terminal domain, the functional importance of which is unknown.MethodsRecombinant hNABP1/2 proteins were purified using affinity and size exclusion chromatography and their identities confirmed by mass spectrometry. Oligomerization state was checked by sucrose gradient centrifugation. Secondary structure was determined by circular dichroism spectroscopy. Nucleic acid binding ability was examined by EMSA and ITC.ResultsBoth hNABP1 and hNABP2 exist as monomers in solution; however, hNABP2 exhibits anomalous behavior. CD spectroscopy revealed that the C-terminus of hNABP2 is highly disordered. Deletion of the C-terminal tail diminishes the DNA binding ability and protein stability of hNABP2. Although both hNABP1 and hNABP2 prefer to bind ssDNA than double-stranded (ds) DNA, hNABP1 has a higher affinity for ssDNA than hNABP2. Unlike hNABP2, hNABP1 protein binds and multimerizes on ssDNA with the C-terminal tail responsible for its multimerization. Both hNABP1 and hNABP2 are able to bind single-stranded RNA, with hNABP2 having a higher affinity than hNABP1.ConclusionsBiochemical evidence suggests that the C-terminal region of NABP1 and NABP2 is essential for their functionality and may lead to different roles in DNA and RNA metabolism.General significanceThis is the first report demonstrating the regulation and functional properties of the C-terminal domain of hNABP1/2, which might be a general characteristic of OB-fold proteins.