V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1-RAG2 ...protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2 Å resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling ...of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recent crystallographic analysis of p66/p51 human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) complexed with a non-polypurine tract RNA/DNA hybrid has illuminated novel and ...important contacts between structural elements at the C terminus of the noncatalytic p51 subunit and the nucleic acid duplex in the vicinity of the ribonuclease H (RNase H) active site. In particular, a short peptide spanning residues Phe-416–Pro-421 was shown to interact with the DNA strand, cross the minor groove of the helix, and then form Van der Waals contacts with the RNA strand adjacent to the scissile phosphate. At the base of the adjoining α-helix M′, Tyr-427 forms a hydrogen bond with Asn-348, the latter of which, when mutated to Ile, is implicated in resistance to both nucleoside and non-nucleoside RT inhibitors. Based on our structural data, we analyzed the role of the p51 C terminus by evaluating selectively mutated p66/p51 heterodimers carrying (i) p51 truncations that encroach on α-M′, (ii) alterations that interrupt the Asn-348-Tyr-427 interaction, and (iii) alanine substitutions throughout the region Phe-416–Pro-421. Collectively, our data support the notion that the p51 C terminus makes an important contribution toward hybrid binding and orienting the RNA strand for catalysis at the RNase H active site.
Background: New crystallographic data for HIV-1 RT containing an RNA/DNA hybrid show important interactions involving the p51 C terminus.
Results: Altered hydrogen bonding at the p51 C terminus affects both NNRTI sensitivity and enzyme activity.
Conclusion: An intact p51 subunit is necessary to faithfully recapitulate activities of the RT heterodimer.
Significance: The extreme C terminus of p51 plays an important role in RNA/DNA hybrid positioning and hydrolysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Type I restriction-modification enzymes act as conventional adenine methylases on hemimethylated DNAs, but unmethylated recognition targets induce them to translocate thousands of base pairs before ...cleaving distant sites nonspecifically. The first crystal structure of a type I motor subunit responsible for translocation and cleavage suggests how the pentameric translocating complex is assembled and provides a structural framework for translocation of duplex DNA by RecA-like ATPase motors.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal ...subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV‐1 that target integrase recognize the ...nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV‐1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV‐1 DNA integration.
PDB Code(s): 4bac
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Mammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 ...protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 paired complex is active in the presence of Mg2+, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex. We report here the purification and initial crystallization of the RAG1/2 signal-end complex for atomic-resolution structure elucidation. Strict pairing of the 12RSS and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.
Background: V(D)J recombination, initiated by the RAG1/2 protein complex, requires two types of DNA recombination signal sequences (RSS), 12RSS and 23RSS.
Results: Improved RAG1/2 expression and purification allowed us to systematically assemble protein-DNA complexes.
Conclusion: RAG1/2 binds one copy each of 12RSS and 23RSS DNA.
Significance: Strictly enforced pairing of the 12RSS and 23RSS explains the recombination specificity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hundreds of structures of type 1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined, but only one contains an RNA/DNA hybrid. Here we report three structures of ...HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid-RT structures including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT-DNA complexes. Thus, the hybrid has ready access to the RNase-H active site. These observations indicate that an RT-nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT-hybrid interface that undergoes conformational changes between two catalytic states.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
V(D)J recombination in the vertebrate immune system generates a highly diverse population of immunoglobulins and T-cell receptors by combinatorial joining of segments of coding DNA. The RAG1- RAG2 ...protein complex initiates this site-specific recombination by cutting DNA at specific sites flanking the coding segments. Here we report the crystal structure of the mouse RAG1-RAG2 complex at 3.2A resolution. The 230-kilodalton RAG1-RAG2 heterotetramer is 'Y-shaped', with the amino-terminal domains of the two RAG1 chains forming an intertwined stalk. Each RAG1-RAG2 heterodimer composes one arm of the 'Y', with the active site in the middle and RAG2 at its tip. The RAG1-RAG2 structure rationalizes more than 60 mutations identified in immunodeficient patients, as well as a large body of genetic and biochemical data. The architectural similarity between RAG1 and the hairpin-forming transposases Hermes and Tn5 suggests the evolutionary conservation of these DNA rearrangements.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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