1ahd: Difference between revisions

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DETERMINATION OF THE NMR SOLUTION STRUCTURE OF AN ANTENNAPEDIA HOMEODOMAIN-DNA COMPLEX

OverviewOverview

The nuclear magnetic resonance (NMR) solution structure of a complex formed by the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp(C39S), and a 14 base-pair DNA duplex containing the BS2 operator sequence was determined using uniform 13C and 15N-labeling of the protein. Two-dimensional nuclear Overhauser enhancement spectroscopy ([1H,1H]NOESY) with 15N(omega 2)-half-filter and 13C(omega 1, omega 2)-double-half-filter, and three-dimensional heteronuclear-correlated [1H,1H]NOESY yielded a total of 855 intramolecular NOE upper distance constraints in the homeodomain, 151 upper distance constraints within the DNA duplex, and 39 intermolecular protein-DNA upper distance constraints. These data were used as the input for the structure calculation with simulated annealing followed by molecular dynamics in a water bath and energy refinement. A group of 16 conformers was thus generated which represent the solution structure of the Antp(C39S) homeodomain-DNA complex. The new structure determination confirms the salient features reported previously from a preliminary investigation of the same complex, in particular the location of the recognition helix in the major groove with the turn of the helix-turn-helix motif outside the contact area with the DNA, and the N-terminal arm of the homeodomain contacting the minor groove of the DNA. In addition, distinct amino acid side-chain-DNA contacts could be identified, and evidence was found that the invariant residue Asn51 (and possibly also Gln50) is in a slow dynamic equilibrium between two or several different DNA contact sites. The molecular dynamics calculations in a water bath yielded structures with hydration water molecules in the protein-DNA interface, which coincides with direct NMR observations of hydration waters. In the Appendix the experimental data obtained with the Antp(C39S) homeodomain-DNA complex and the techniques used for the structure calculation are evaluated using a simulated input data set derived from the X-ray crystal structure of a DNA complex with a homologous homeodomain. This study indicates that a nearly complete set of NOE upper distance constraints for the Antp(C39S) homeodomain and the protein-DNA interface was presently obtained. It further shows that the structure calculation used here yields a precise reproduction of the crystal structure from the simulated input data, and also results in hydration of the protein-DNA interface in the recalculated complex.

About this StructureAbout this Structure

1AHD is a Single protein structure of sequence from Drosophila melanogaster. Full crystallographic information is available from OCA.

ReferenceReference

Determination of the nuclear magnetic resonance solution structure of an Antennapedia homeodomain-DNA complex., Billeter M, Qian YQ, Otting G, Muller M, Gehring W, Wuthrich K, J Mol Biol. 1993 Dec 20;234(4):1084-93. PMID:7903398

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-[[Image:1ahd.gif|left|200px]]<br /><applet load="1ahd" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1ahd.gif|left|200px]]<br /><applet load="1ahd" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1ahd" />
 '''DETERMINATION OF THE NMR SOLUTION STRUCTURE OF AN ANTENNAPEDIA HOMEODOMAIN-DNA COMPLEX'''<br />
 ==Overview==
-The nuclear magnetic resonance (NMR) solution structure of a complex, formed by the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp(C39S), and a 14 base-pair DNA duplex containing the BS2 operator, sequence was determined using uniform 13C and 15N-labeling of the protein., Two-dimensional nuclear Overhauser enhancement spectroscopy ([1H,1H]NOESY), with 15N(omega 2)-half-filter and 13C(omega 1, omega, 2)-double-half-filter, and three-dimensional heteronuclear-correlated, [1H,1H]NOESY yielded a total of 855 intramolecular NOE upper distance, constraints in the homeodomain, 151 upper distance constraints within the, DNA duplex, and 39 intermolecular protein-DNA upper distance constraints., These data were used as the input for the structure calculation with, simulated annealing followed by molecular dynamics in a water bath and, energy refinement. A group of 16 conformers was thus generated which, represent the solution structure of the Antp(C39S) homeodomain-DNA, complex. The new structure determination confirms the salient features, reported previously from a preliminary investigation of the same complex, in particular the location of the recognition helix in the major groove, with the turn of the helix-turn-helix motif outside the contact area with, the DNA, and the N-terminal arm of the homeodomain contacting the minor, groove of the DNA. In addition, distinct amino acid side-chain-DNA, contacts could be identified, and evidence was found that the invariant, residue Asn51 (and possibly also Gln50) is in a slow dynamic equilibrium, between two or several different DNA contact sites. The molecular dynamics, calculations in a water bath yielded structures with hydration water, molecules in the protein-DNA interface, which coincides with direct NMR, observations of hydration waters. In the Appendix the experimental data, obtained with the Antp(C39S) homeodomain-DNA complex and the techniques, used for the structure calculation are evaluated using a simulated input, data set derived from the X-ray crystal structure of a DNA complex with a, homologous homeodomain. This study indicates that a nearly complete set of, NOE upper distance constraints for the Antp(C39S) homeodomain and the, protein-DNA interface was presently obtained. It further shows that the, structure calculation used here yields a precise reproduction of the, crystal structure from the simulated input data, and also results in, hydration of the protein-DNA interface in the recalculated complex.
+The nuclear magnetic resonance (NMR) solution structure of a complex formed by the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp(C39S), and a 14 base-pair DNA duplex containing the BS2 operator sequence was determined using uniform 13C and 15N-labeling of the protein. Two-dimensional nuclear Overhauser enhancement spectroscopy ([1H,1H]NOESY) with 15N(omega 2)-half-filter and 13C(omega 1, omega 2)-double-half-filter, and three-dimensional heteronuclear-correlated [1H,1H]NOESY yielded a total of 855 intramolecular NOE upper distance constraints in the homeodomain, 151 upper distance constraints within the DNA duplex, and 39 intermolecular protein-DNA upper distance constraints. These data were used as the input for the structure calculation with simulated annealing followed by molecular dynamics in a water bath and energy refinement. A group of 16 conformers was thus generated which represent the solution structure of the Antp(C39S) homeodomain-DNA complex. The new structure determination confirms the salient features reported previously from a preliminary investigation of the same complex, in particular the location of the recognition helix in the major groove with the turn of the helix-turn-helix motif outside the contact area with the DNA, and the N-terminal arm of the homeodomain contacting the minor groove of the DNA. In addition, distinct amino acid side-chain-DNA contacts could be identified, and evidence was found that the invariant residue Asn51 (and possibly also Gln50) is in a slow dynamic equilibrium between two or several different DNA contact sites. The molecular dynamics calculations in a water bath yielded structures with hydration water molecules in the protein-DNA interface, which coincides with direct NMR observations of hydration waters. In the Appendix the experimental data obtained with the Antp(C39S) homeodomain-DNA complex and the techniques used for the structure calculation are evaluated using a simulated input data set derived from the X-ray crystal structure of a DNA complex with a homologous homeodomain. This study indicates that a nearly complete set of NOE upper distance constraints for the Antp(C39S) homeodomain and the protein-DNA interface was presently obtained. It further shows that the structure calculation used here yields a precise reproduction of the crystal structure from the simulated input data, and also results in hydration of the protein-DNA interface in the recalculated complex.
 ==About this Structure==
-AHD is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Drosophila_melanogaster Drosophila melanogaster]. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1AHD OCA].
+AHD is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Drosophila_melanogaster Drosophila melanogaster]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1AHD OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Single protein]]
 [[Category: Billeter, M.]]
-[[Category: Gehring, W.J.]]
+[[Category: Gehring, W J.]]
 [[Category: Muller, M.]]
 [[Category: Otting, G.]]
-[[Category: Qian, Y.Q.]]
+[[Category: Qian, Y Q.]]
 [[Category: Wuthrich, K.]]
 [[Category: dna binding protein/dna]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 10:51:16 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 11:44:29 2008''