NMR Structure of the Amino-Terminal Domain of the Lambda Integrase Protein in Complex with DNA: Immobilization of a Flexible Tail Facilitates Beta-Sheet Recognition of the Major Groove

The integrase protein (Int) from bacteriophage lambda is the archetypal member of the tyrosine recombinase family, a large group of enzymes that rearrange DNA in all domains of life. Int catalyzes the insertion and excision of the viral genome into and out of the Escherichia coli chromosome. Recombination transpires within higher-order nucleoprotein complexes that form when its amino-terminal domain binds to arm-type DNA sequences that are located distal to the site of strand exchange. Arm-site binding by Int is essential for catalysis, as it promotes Int-mediated bridge structures that stabilize the recombination machinery. We have elucidated how Int is able to sequence specifically recognize the arm-type site sequence by determining the solution structure of its amino-terminal domain (Int^N, residues Met1 to Leu64) in complex with its P′2 DNA binding site. Previous studies have shown that Int^N adopts a rare monomeric DNA binding fold that consists of a three-stranded antiparallel beta-sheet that is packed against a carboxy-terminal alpha helix. A low-resolution crystal structure of the full-length protein also revealed that the sheet is inserted into the major groove of the arm-type site. The solution structure presented here reveals how Int^N specifically recognizes the arm-type site sequence. A novel feature of the new solution structure is the use of an 11-residue tail that is located at the amino terminus. DNA binding induces the folding of a 3₁₀ helix in the tail that projects the amino terminus of the protein deep into the minor groove for stabilizing DNA contacts. This finding reveals the structural basis for the observation that the “unstructured” amino terminus is required for recombination.     

Crystal Structure of DNA Cytidine Deaminase ABOBEC3G Catalytic Deamination Domain Suggests a Binding Mode of Full-length Enzyme to Single-stranded DNA

APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA.     

The N-terminal extensions of Deinococcus radiodurans Dps-1 mediate DNA major groove interactions as well as assembly of the dodecamer

Dps (DNA protection during starvation) proteins play an important role in the protection of prokaryotic macromolecules from damage by reactive oxygen species. Previous studies have suggested that the lysine-rich N-terminal tail of Dps proteins participates in DNA binding. In comparison with other Dps proteins, Dps-1 from Deinococcus radiodurans has an extended N terminus comprising 55 amino acids preceding the first helix of the 4-helix bundle monomer. In the crystal structure of Dps-1, the first approximately 30 N-terminal residues are invisible, and the remaining 25 residues form a loop that harbors a novel metal-binding site. We show here that deletion of the flexible N-terminal tail obliterates DNA/Dps-1 interaction. Surprisingly, deletion of the entire N terminus also abolishes dodecameric assembly of the protein. Retention of the N-terminal metal site is necessary for formation of the dodecamer, and metal binding at this site facilitates oligomerization of the protein. Electrophoretic mobility shift assays using DNA modified with specific major/minor groove reagents further show that Dps-1 interacts through the DNA major groove. DNA cyclization assays suggest that dodecameric Dps-1 does not wrap DNA about itself. A significant decrease in DNA binding affinity accompanies a reduction in duplex length from 22 to 18 bp, but only for dodecameric Dps-1. Our data further suggest that high affinity DNA binding depends on occupancy of the N-terminal metal site. Taken together, the mode of DNA interaction by dodecameric Dps-1 suggests interaction of two metal-anchored N-terminal tails in successive DNA major grooves, leading to DNA compaction by formation of stacked protein-DNA layers.     

Using Soft X-Rays for a Detailed Picture of Divalent Metal Binding in the Nucleosome

Divalent metals associate with DNA in a site-selective manner, which can influence nucleosome positioning, mobility, compaction, and recognition by nuclear factors. We previously characterized divalent metal binding in the nucleosome core using hard (short-wavelength) X-rays allowing high-resolution crystallographic determination of the strongest affinity sites, which revealed that Mn²⁺ associates with the DNA major groove in a sequence- and conformation-dependent manner. In this study, we obtained diffraction data with soft X-rays at the Mn²⁺ absorption edge for a core particle crystal in the presence of 10 mM MnSO₄, mimicking prevailing Mg²⁺ concentration in the nucleus. This provides an exceptional view of counterion binding in the nucleosome through identification of 45 divalent metal binding sites.In addition to that at the well-characterized major interparticle interface, only one other histone-divalent metal binding site is found, which corresponds to a symmetry-related counterpart on the ‘free’ H2B α1 helix C-terminus. This emphasizes the importance of the α-helix dipole in ion binding and suggests that the H2B motif may serve as a nucleation site in nucleosome compaction. The 43 sites associated with the DNA are characterized by (1) high-affinity direct coordination at the most electrostatically favorable major groove locations, (2) metal hydrate binding to the major groove, (3) direct coordination to phosphate groups at sites of high charge density, (4) metal hydrate binding in the minor groove, or (5) metal hydrate-divalent anion pairing. Metal hydrates are found within the minor groove only at locations displaying a narrow range of high-intermediate width and to which histone N-terminal tails are not associated or proximal. This indicates that divalent metals and histone tails can both collaborate and compete in minor groove association, which sheds light on nucleosome solubility and chromatin compaction behavior.     

Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA

Coronavirus nucleocapsid proteins are basic proteins that encapsulate viral genomic RNA to form part of the virus structure. The nucleocapsid protein of SARS-CoV is highly antigenic and associated with several host-cell interactions. Our previous studies using nuclear magnetic resonance revealed the domain organization of the SARS-CoV nucleocapsid protein. RNA has been shown to bind to the N-terminal domain (NTD), although recently the C-terminal half of the protein has also been implicated in RNA binding. Here, we report that the C-terminal domain (CTD), spanning residues 248-365 (NP248-365), had stronger nucleic acid-binding activity than the NTD. To determine the molecular basis of this activity, we have also solved the crystal structure of the NP248-365 region. Residues 248-280 form a positively charged groove similar to that found in the infectious bronchitis virus (IBV) nucleocapsid protein. Furthermore, the positively charged surface area is larger in the SARS-CoV construct than in the IBV. Interactions between residues 248-280 and the rest of the molecule also stabilize the formation of an octamer in the asymmetric unit. Packing of the octamers in the crystal forms two parallel, basic helical grooves, which may be oligonucleotide attachment sites, and suggests a mechanism for helical RNA packaging in the virus.     

含磷酸结合口袋的BRCT结构域功能位点预测

BRCT( BRCA1 C-terminus)是真核生物DNA损伤修复系统重要的信号传导和蛋白靶向结构域.为了探讨含磷酸结合口袋的BRCT与磷酸化配体结合的机制,对XRCC1 BRCT1、PTIP BRCT4、ECT2 BRCT1和TopBP1BRCT1进行了结构保守性和表面静电势分析.结果显示,4个BRCT的磷酸结合口袋周围所存在的结构保守并带正电势的沟槽很可能是其功能位点,并且类似的沟槽在含磷酸结合口袋的BRCT中普遍存在.沟槽两侧及底部均带有极性氨基酸残基,两侧带正电荷,而底部疏水.这说明沟槽与配体的结合以静电和疏水相互作用为主.沟槽主要位于单个BRCT中,而且4个BRCT的沟槽在形状和电荷分布上都不同,说确明BRCT配体特异性主要由单个BRCT决定.磷酸结合口袋位于沟槽中心,说明沟槽可能同时结合磷酸化残基的N端和C端附近残基.     

NMR assignments for the amino-terminal residues of trp repressor and their role in DNA binding

The trp repressor of Escherichia coli specifically binds to operator DNAs in three operons involved in tryptophan metabolism. The NMR spectra of repressor and a chymotryptic fragment lacking the six amino-terminal residues are compared. Two-dimensional J-correlated spectra of the two forms of the protein are superimposable except for cross-peaks that are associated with the N-terminal region. The chemical shifts and relaxation behavior of the N-terminal resonances suggest mobile "arms". Spin-echo experiments on a ternary complex of repressor with L-tryptophan and operator DNA indicate that the termini are also disordered in the complex, although removal of the arms reduces the DNA binding energy. Relaxation measurements on the armless protein show increased mobility for several residues, probably due to helix fraying in the newly exposed N-terminal region. DNA binding by the armless protein does not reduce the mobility of these residues. Thus, it appears that the arms serve to stabilize the N-terminal helix but that this structural role does not explain their contribution to the DNA binding energy. These results suggest that the promiscuous DNA binding by the arms seen in the X-ray crystal structure is found in solution as well.     

Structural implications into dsRNA binding and RNA silencing suppression by NS3 protein of Rice Hoja Blanca Tenuivirus

Rice Hoja Blanca Tenuivirus (RHBV), a negative strand RNA virus, has been identified to infect rice and is widely transmitted by the insect vector. NS3 protein encoded by RHBV RNA3 was reported to be a potent RNAi suppressor to counterdefense RNA silencing in plants, insect cells, and mammalian cells. Here, we report the crystal structure of the N-terminal domain of RHBV NS3 (residues 21–114) at 2.0 Å. RHBV NS3 N-terminal domain forms a dimer by two pairs of α-helices in an anti-parallel mode, with one surface harboring a shallow groove at the dimension of 20 Å × 30 Å for putative dsRNA binding. In vitro RNA binding assay and RNA silencing suppression assay have demonstrated that the structural conserved residues located along this shallow groove, such as Arg50, His51, Lys77, and His85, participate in dsRNA binding and RNA silencing suppression. Our results provide the initial structural implications in understanding the RNAi suppression mechanism by RHBV NS3.     

DNA recognition by the brinker repressor--an extreme case of coupling between binding and folding

The Brinker (Brk) nuclear repressor is a major element of the Drosophila Decapentaplegic morphogen signaling pathway. Its N-terminal part has weak homology to the Antennapedia homeodomain and binds to GC-rich DNA sequences. We have investigated the conformation and dynamics of the N-terminal 101 amino acid residues of Brk in the absence and in the presence of cognate DNA by solution NMR spectroscopy. In the absence of DNA, Brk is unfolded and highly flexible throughout the entire backbone. Addition of cognate DNA induces the formation of a well-folded structure for residues R46 to R95. This structure consists of four helices forming a helix-turn-helix motif that differs from homeodomains, but has similarities to the Tc3 transposase, the Pax-6 Paired domain, and the human centromere-binding protein. The GC-rich DNA recognition can be explained by specific major groove hydrogen bonds from the N-terminal end of helix alpha3. The transition from a highly flexible, completely unfolded conformation in the absence of DNA to a well-formed structure in the complex presents a very extreme case of the "coupling of binding and folding" phenomenon.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

Oxidation-sensitive residues mediate the DNA bending abilities of the architectural MC1 protein

The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially. We show by site directed mutagenesis that two of these residues, Trp74 and Met75, are involved in the DNA binding. Their substitution by alanine leads to a strong reduction in the protein capacity to bend DNA, and a total loss of its ability to recognize bent DNA. Taken together, these results show that oxidation of both these residues is responsible for the protein inactivation. Furthermore, the results confirm the strong relationship between DNA bending and recognition of DNA sequences by the MC1 protein.     

NMR structure of the N-terminal domain of SUMO ligase PIAS1 and its interaction with tumor suppressor p53 and A/T-rich DNA oligomers

A member of the PIAS (protein inhibitor of activated STAT) family of proteins, PIAS1, have been reported to serve as an E3-type SUMO ligase for tumor suppressor p53 and its own. It also was proposed that the N-terminal domain of PIAS1 interacts with DNA as well as p53. Extensive biochemical studies have been devoted recently to understand sumoylations and its biological implications, whereas the structural aspects of the PIAS family and the mechanism of its interactions with various factors are less well known to date. In this study, the three-dimensional structure of the N-terminal domain (residues 1-65) of SUMO ligase PIAS1 was determined by NMR spectroscopy. The structure revealed a unique four-helix bundle with a topology of an up-down-extended loop-down-up, a part of which the helix-extended loop-helix represented the SAP (SAF-A/B, Acinus, and PIAS) motif. Thus, this N-terminal domain may be referred to as a four-helix SAP domain. The glutathione S-transferase pull-down assay demonstrated that this domain possesses a binding ability to tumor suppressor p53, a target protein for sumoylation by PIAS1, whereas gel mobility assays showed that it has a strong affinity toward A/T-rich DNA. An NMR analysis of the four-helix SAP domain complexed with the 16-bp-long DNA demonstrated that one end of the four-helix bundle is the binding site and may fit into the minor groove of DNA. The three-dimensional structure and its binding duality are discussed in conjunction with the biological functions of PIAS1 as a SUMO ligase.