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Kathryn R Ely - One of the best experts on this subject based on the ideXlab platform.
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new insights on dna recognition by ets proteins from the crystal structure of the pu 1 ets domain dna complex
Journal of Biological Chemistry, 1996Co-Authors: Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Ramadurgam Kodandapani, William Shepard, Kathryn R ElyAbstract:Transcription factors belonging to the ets family regulate gene expression and share a conserved ETS DNA-binding domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The domain is similar to alpha+beta ("winged") Helix-Turn-Helix DNA-binding proteins. The crystal structure of the PU.1 ETS domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ets proteins confirms this complex as a paradigm for ets DNA recognition. The second helix in the Helix-Turn-Helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.
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a new pattern for helix turn helix recognition revealed by the pu 1 ets domain dna complex
Nature, 1996Co-Authors: Ramadurgam Kodandapani, Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Gennaro Piccialli, Kathryn R ElyAbstract:THE Ets family of transcription factors, of which there are now about 35 members1,2, regulate gene expression during growth and development. They share a conserved domain of around 85 amino acids3 which binds as a monomer to the DNA sequence 5'-C/ AGGAA/T-3'. We have determined theocrystal structure of an ETS domain complexed with DNA, at 2.3-A resolution. The domain is similar to α+β (winged) 'helix–turn–helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8°. The domain contacts the DNA by a novel loop–helix–loop architecture. Four of the amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix–turn–helix' motif, which binds downstream and on the opposite strand.
Richard A Maki - One of the best experts on this subject based on the ideXlab platform.
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new insights on dna recognition by ets proteins from the crystal structure of the pu 1 ets domain dna complex
Journal of Biological Chemistry, 1996Co-Authors: Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Ramadurgam Kodandapani, William Shepard, Kathryn R ElyAbstract:Transcription factors belonging to the ets family regulate gene expression and share a conserved ETS DNA-binding domain that binds to the core sequence 5'-(C/A)GGA(A/T)-3'. The domain is similar to alpha+beta ("winged") Helix-Turn-Helix DNA-binding proteins. The crystal structure of the PU.1 ETS domain complexed to a 16-base pair oligonucleotide revealed a pattern for DNA recognition from a novel loop-helix-loop architecture (Kodandapani, R., Pio, F., Ni. C.-Z., Piccialli, G., Klemsz, M., McKercher, S., Maki, R. A., and Ely, K. R. (1996) Nature 380, 456-460). Correlation of this model with mutational analyses and chemical shift data on other ets proteins confirms this complex as a paradigm for ets DNA recognition. The second helix in the Helix-Turn-Helix motif lies deep in the major groove with specific contacts with bases in both strands in the core sequence made by conserved residues in alpha3. On either side of this helix, two loops contact the phosphate backbone. The DNA is bent (8 degrees) but uniformly curved without distinct kinks. ETS domains bind DNA as a monomer yet make extensive DNA contacts over 30 A. DNA bending likely results from phosphate neutralization of the phosphate backbone in the minor groove by both loops in the loop-helix-loop motif. Contacts from these loops stabilize DNA bending and may mediate specific base interactions by inducing a bend toward the protein.
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a new pattern for helix turn helix recognition revealed by the pu 1 ets domain dna complex
Nature, 1996Co-Authors: Ramadurgam Kodandapani, Frederic Pio, Scott R Mckercher, Michael J Klemsz, Richard A Maki, Gennaro Piccialli, Kathryn R ElyAbstract:THE Ets family of transcription factors, of which there are now about 35 members1,2, regulate gene expression during growth and development. They share a conserved domain of around 85 amino acids3 which binds as a monomer to the DNA sequence 5'-C/ AGGAA/T-3'. We have determined theocrystal structure of an ETS domain complexed with DNA, at 2.3-A resolution. The domain is similar to α+β (winged) 'helix–turn–helix' proteins and interacts with a ten-base-pair region of duplex DNA which takes up a uniform curve of 8°. The domain contacts the DNA by a novel loop–helix–loop architecture. Four of the amino acids that directly interact with the DNA are highly conserved: two arginines from the recognition helix lying in the major groove, one lysine from the 'wing' that binds upstream of the core GGAA sequence, and another lysine, from the 'turn' of the 'helix–turn–helix' motif, which binds downstream and on the opposite strand.
Chen Lin - One of the best experts on this subject based on the ideXlab platform.
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A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)
Journal of Biomedical Science, 2012Co-Authors: Po-tsang Huang, Kuo-long Lou, Chen LinAbstract:Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family. Conclusions The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.
Alan Herbert - One of the best experts on this subject based on the ideXlab platform.
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structure function analysis of the z dna binding domain zalpha of dsrna adenosine deaminase type i reveals similarity to the alpha beta family of helix turn helix proteins
The EMBO Journal, 1999Co-Authors: Markus Schade, Christopher J Turner, Ky Lowenhaupt, Alexander Rich, Alan HerbertAbstract:RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins. An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zalpha and Zbeta, the function of which is as yet unknown. In this study, multidimensional NMR spectroscopy was used to show that the topology of Zalpha is alpha1beta1alpha2alpha3beta2beta3. Long-range NOEs indicate that beta1 and beta3 interact with each other. Site-directed mutagenesis was used to identify residues in alpha3, beta3 and the loop connecting beta2 to beta3 that affect Z-DNA binding. Also identified were 11 hydrophobic residues that are essential for protein stability. Comparison with known structures reveals some similarity between Zalpha and (alpha + beta) Helix-Turn-Helix proteins, such as histone 5 and the family of hepatocyte nuclear factor-3 winged-Helix-Turn-Helix transcription factors. Taken together, the structural and functional data suggest that recognition of Z-DNA by Zalpha involves residues in both the alpha3 helix and the C-terminal beta-sheet.
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structure function analysis of the z dna binding domain zalpha of dsrna adenosine deaminase type i reveals similarity to the alpha beta family of helix turn helix proteins
The EMBO Journal, 1999Co-Authors: Markus Schade, Christopher J Turner, Ky Lowenhaupt, Alexander Rich, Alan HerbertAbstract:RNA editing alters pre-mRNA through site-selective adenosine deamination, which results in codon changes that lead to the production of novel proteins. An enzyme that catalyzes this reaction, double-stranded RNA adenosine deaminase (ADAR1), contains two N-terminal Z-DNA-binding motifs, Zα and Zβ, the function of which is as yet unknown. In this study, multidimensional NMR spectroscopy was used to show that the topology of Zα is α1β1α2α3β2β3. Long-range NOEs indicate that β1 and β3 interact with each other. Site-directed mutagenesis was used to identify residues in α3, β3 and the loop connecting β2 to β3 that affect Z-DNA binding. Also identified were 11 hydrophobic residues that are essential for protein stability. Comparison with known structures reveals some similarity between Zα and (α + β) helix–turn–helix proteins, such as histone 5 and the family of hepatocyte nuclear factor-3 winged-helix–turn–helix transcription factors. Taken together, the structural and functional data suggest that recognition of Z-DNA by Zα involves residues in both the α3 helix and the C-terminal β-sheet.
Po-tsang Huang - One of the best experts on this subject based on the ideXlab platform.
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A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)
Journal of Biomedical Science, 2012Co-Authors: Po-tsang Huang, Kuo-long Lou, Chen LinAbstract:Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family. Conclusions The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.