Effects of dimer interface mutations in Hin recombinase on DNA binding and recombination

2001 ◽  
Vol 266 (4) ◽  
pp. 598-607 ◽  
Author(s):  
H. Lee ◽  
S. Lee ◽  
H. Lee ◽  
H. Lim
Keyword(s):  
Dna Binding ◽  
Dimer Interface ◽  
Hin Recombinase

scholarly journals Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

2003 ◽  
Vol 185 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Yinghua Chen ◽  
Catherine Birck ◽  
Jean-Pierre Samama ◽  
F. Marion Hulett
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Salt Bridge ◽  
Pho Regulon ◽  
Dimer Interface ◽  
And Function ◽  
Negative Phenotype

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

Crystallization and Preliminary X-ray Analysis of the DNA Binding Domain of the Hin Recombinase with Its DNA Binding Site

1993 ◽  
Vol 232 (3) ◽  
pp. 982-986
Author(s):  
Jin-An Feng ◽  
Melvin Simon ◽  
David P. Mack ◽  
Peter B. Dervan ◽  
Reid C. Johnson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Site ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
X Ray ◽  
Dna Binding Site ◽  
Hin Recombinase

scholarly journals Hin Recombinase Mutants Functionally Disrupted in Interactions with Fis

2001 ◽  
Vol 183 (1) ◽  
pp. 28-35 ◽  
Author(s):  
Oliver Z. Nanassy ◽  
Kelly T. Hughes
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Genetic Screen ◽  
Recombination Reaction ◽  
Genetic Classification ◽  
Hin Recombinase

ABSTRACT A previous genetic screen was designed to separate Hin recombinase mutants into distinct classes based on the stage in the recombination reaction at which they are blocked (O. Nanassy, Zoltan, and K. T. Hughes, Genetics 149:1649–1663, 1998). One class of DNA binding-proficient, recombination-deficient mutants was predicted by genetic classification to be defective in the step prior to invertasome formation. Based on the genetic criteria, mutants from this class were also inferred to be defective in interactions with Fis. In order to understand how the genetic classification relates to individual biochemical steps in the recombination reaction these mutants, R123Q, T124I, and A126T, were purified and characterized for DNA cleavage and recombination activities. Both the T124I and A126T mutants were partially active, whereas the R123Q mutant was inactive. The A126T mutant was not as defective for recombination as the T124I allele and could be partially rescued for recombination both in vivo and in vitro by increasing the concentration of Fis protein. Rescue of the A126T allele required the Fis protein to be DNA binding proficient. A model for a postsynaptic role for Fis in the inversion reaction is presented.

scholarly journals DNA-binding properties of the Hin recombinase

1989 ◽  
Vol 264 (17) ◽  
pp. 10072-10082
Author(s):  
A C Glasgow ◽  
M F Bruist ◽  
M I Simon
Keyword(s):  
Dna Binding ◽  
Binding Properties ◽  
Hin Recombinase ◽  
Dna Binding Properties

scholarly journals Two crystal structures reveal design for repurposing the C-Ala domain of human AlaRS

2016 ◽  
Vol 113 (50) ◽  
pp. 14300-14305 ◽  
Author(s):  
Litao Sun ◽  
Youngzee Song ◽  
David Blocquel ◽  
Xiang-Lei Yang ◽  
Paul Schimmel
Keyword(s):  
Dna Binding ◽  
Crystal Structures ◽  
Sequence Divergence ◽  
Dimer Interface ◽  
Trna Synthetases ◽  
X Ray Scattering ◽  
Aminoacyl Trna Synthetases ◽  
Single Exception ◽  
Binding Groove

The 20 aminoacyl tRNA synthetases (aaRSs) couple each amino acid to their cognate tRNAs. During evolution, 19 aaRSs expanded by acquiring novel noncatalytic appended domains, which are absent from bacteria and many lower eukaryotes but confer extracellular and nuclear functions in higher organisms. AlaRS is the single exception, with an appended C-terminal domain (C-Ala) that is conserved from prokaryotes to humans but with a wide sequence divergence. In human cells, C-Ala is also a splice variant of AlaRS. Crystal structures of two forms of human C-Ala, and small-angle X-ray scattering of AlaRS, showed that the large sequence divergence of human C-Ala reshaped C-Ala in a way that changed the global architecture of AlaRS. This reshaping removes the role of C-Ala in prokaryotes for docking tRNA and instead repurposes it to form a dimer interface presenting a DNA-binding groove. This groove cannot form with the bacterial ortholog. Direct DNA binding by human C-Ala, but not by bacterial C-Ala, was demonstrated. Thus, instead of acquiring a novel appended domain like other human aaRSs, which engendered novel functions, a new AlaRS architecture was created by diversifying a preexisting appended domain.

scholarly journals Mutual Information Identifies Sequence Positions Conserved within the Nuclear Receptor Superfamily: Approach Reveals Functionally Important Regions for DNA Binding Specificity

2011 ◽  
Vol 9 (1) ◽  
pp. nrs.09001 ◽  
Author(s):  
Scooter Willis ◽  
Patrick Griffin
Keyword(s):  
Dna Binding ◽  
Mutual Information ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Binding Specificity ◽  
Oligomeric State ◽  
Dimer Interface ◽  
Nuclear Receptor Superfamily ◽  
Dna Binding Specificity ◽  
Flanking Region

Members of the nuclear receptor superfamily differentiate in terms of specificity for DNA recognition and binding, oligomeric state, and ligand binding. The wide range of specificities are impressive given the high degree of sequence conservation in the DNA binding domain (DBD) and moderate sequence conservation with high structural similarity within the ligand binding domains (LBDs). Determining sequence positions that are conserved within nuclear receptor subfamilies can provide important indicators into the structural dynamics that translate to oligomeric state of the active receptor, DNA binding specificity and ligand affinity and selectivity. Here we present a method to analyze sequence data from all nuclear receptors that facilitates detection of co-evolving pairs using Mutual Information (MI). Using this method we demonstrate that MI can reveal functionally important sequence positions within the superfamily and the approach identified three sequence positions that have conserved sequence patterns across all nuclear receptors and subfamilies. Interestingly, two of the sequence positions identified are located within the DBD CII and the third was within Helix c of the DBD. These sequences are located within the heterodimer interface of PPARγ (CII) and RXR α (Helix c) based on PDB:3DZU. Helix c of PPARγ, which is not involved in the DBD dimer interface, binds the minor groove in the 5’ flanking region in a consensus PPARγ response element (PPRE) and the corresponding RXR α (CII) is found in the 3’ flanking region of RXRE (3DZU). As these three sequence positions represent unique identifiers for all nuclear receptors and they are located within the dimer interface of PPARγ-RXRα DBD (3DZU) interfacing with the flanking regions of the NRRE, we conclude they are critical sequence positions perhaps dictating nuclear receptor (NR) DNA binding specificity.

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