Determination of the structure of the DNA binding domain of γ^Δ resolvase in solution

Abstract

The DNA binding domain (DBD) of y6 resolvase (residues 141-183) is responsible for the interaction of this sitespecific DNA recombinase with consensus site DNA within the y6 transposable element in Escherichia coli. Based on chemical-shift comparisons, the proteolytically isolated DBD displays side-chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, JBiol Chem 268:16309-16315). The structure of the DBD in solution has been determined using restraints obtained from 2-dimensional proton NMR data and is represented by 17 conformers. Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 $ and x1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data.With respect to the computed average structure, these conformers display an RMS deviation of 0.67 A for the heavy backbone atoms and 1.49 A for all heavy atoms within residues 149-180. The DBD consists of 3 a-helices comprising residues D149-Q157, S162-T167, and R172-N183. Helix3 and helix-3 form a backbone fold, which is similar to the canonical helix-turn-helix motif. The conformation of the NH2-terminaI residues, G141-R148, appears flexible in solution. A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns. Helix-1 and helix-3 cross with a right-handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix-3 in the major groove and the amino-terminal arm in the minor groove. This structure represents an important step toward analysis of the mechanism of DNA interaction by y6 resolvase and provides initial structure-function comparisons among the divergent DBDs of related resolvases and invertases.Keywords: DNA recombinase; helix-turn-helix; NMR; recognition helix y6 Resolvase is representative of a family of DNA recombinases for which significant structural and functional information regarding site-specific DNA recombination has been obtained. However, until recently, no structural information had been available on the DNA binding domains for this protein family.The y6 resolvase protein, through formation of a multimeric complex termed a synaptosome, catalyzes the second step of y6 DNA transposition in Escherichia coli (reviewed by . In this step, a negatively supercoiled plasmid DNA containing 2 copies of the y6 transposon is converted to catenated circular DNA products, each of which contains a single copy of the transposon. The topolog-

Structural Classification of HTH DNA-binding Domains and Protein – DNA Interaction Modes

“…The codes that end with an X were kindly supplied by their authors. The references of the structures are 1ADR (Sevilla-Sierra et al, 1994); 1BIA (Wilson et al, 1992); 1CGP (Schultz et al, 1991); 1DTR (Qiu et al, 1995); 1LCC (Chuprina et al, 1993); 1LEB ; 1LMB (Beamer & Pabo, 1992); 1PNR (Schumacher et al, 1994); 1R69 (Mondragon et al, 1989a); 2OR1 (Aggarwal et al, 1988); 2CRO (Mondragon et al, 1989b); 3CRO (Mondragon & Harrison, 1991); 3WRP ; 1TRO ; 1FIA (Kostrewa et al, 1992); 1HCR (Feng et al, 1994);1NER (Strzelechka et al, 1995); 1RET (Liu et al, 1994); 1TNT (Clubb et al, 1994); 1APL (Wolberger et al, 1991); 1HDD (Kissinger et al, 1990); 1HOM (Billeter et al, 1990); 1AHD (Billeter et al, 1993); 1LFB (Ceska et al, 1993); 1PHDX (Wilson et al, 1995); 1NK2X (Tsao et al, 1995); 1OCT (Klemm et al, 1994); 1HKT (Vuister et al, 1994); 2HTS (Harrison et al, 1994); 1FLI (Liang et al, 1994); 1HNFX ; 1MSF (Ogata et al, 1994); 1PDN (Xu et al, 1995); 1GHC (Cerf et al, 1994); 1HST (Ramakrishnan et al, 1993).…”

The Serine Recombinases

“…Further insights have come from the structures of the "attachments" to the SR domain. Table 1 summarizes the crystallographic and nuclear magnetic resonance (NMR) structural data on serine recombinases available at the time of submission of this review (45,46,47,48,49,50,51,52). Small-angle scattering-based methods have also provided important structural information (53).…”

Polypeptides Folding: Rules for the Calculation of the Backbone Dihedral Angles φ starting from the Amino Acid Sequence

“…At this purpose, this work has been realized with the aim to define univocally the twisting constants in order to allow, in the end, the precise calculation of the backbone dihedral angles, specifically the backbone dihedral angles φ. With the rules proposed on the twisting constants, as well as on the threshold distances for the chemical interactions, the backbone dihedral angles φ of 1res have been calculated and then compared to the values experimentally obtained by NMR [7] . A comparison between the two sets of data is shown in Figure 2 and can be seen that 38 calculated values, out of 42, show a good agreement with the experimental data [7] , whereas only 2 values have a weak agreement (φ18 and φ25) and just 2 calculated values (φ21 and φ42) do not match the experimental data.…”

“…With the rules proposed on the twisting constants, as well as on the threshold distances for the chemical interactions, the backbone dihedral angles φ of 1res have been calculated and then compared to the values experimentally obtained by NMR [7] . A comparison between the two sets of data is shown in Figure 2 and can be seen that 38 calculated values, out of 42, show a good agreement with the experimental data [7] , whereas only 2 values have a weak agreement (φ18 and φ25) and just 2 calculated values (φ21 and φ42) do not match the experimental data. This results make the method proposed a good tool to determine and predict at least the backbone dihedral angles φ of polypeptides, even if, further improvements will be realized studying other polypeptides.…”