12 research outputs found
Solution structure of the DNA-binding domain of human telomeric protein, hTRF1
AbstractBackground: Mammalian telomeres consist of long tandem arrays of the double-stranded TTAGGG sequence motif packaged by a telomere repeat binding factor, TRF1. The DNA-binding domain of TRF1 shows sequence homology to each of three tandem repeats of the DNA-binding domain of the transcriptional activator c-Myb. The isolated c-Myb-like domain of human TRF1 (hTRF1) binds specifically to telomeric DNA as a monomer, in a similar manner to that of homeodomains. So far, the only three-dimensional structure of a telomeric protein to be determined is that of a yeast telomeric protein, Rap1p. The DNA-binding domain of Rap1p contains two subdomains that are structurally closely related to c-Myb repeats. We set out to determine the solution structure of the DNA-binding domain of hTRF1 in order to establish its mode of DNA binding.Results: The solution structure of the DNA-binding domain of hTRF1 has been determined and shown to comprise three helices. The architecture of the three helices is very similar to that of each Rap1p subdomain and also to that of each c-Myb repeat. The second and third helix form a helix-turn-helix (HTH) variant. The length of the third helix of hTRF1 is similar to that of the second subdomain of Rap1p.Conclusions: The hTRF1 DNA-binding domain is likely to bind to DNA in a similar manner to that of the second subdomain of Rap1p. On the basis of the Rap1p–DNA complex, a model of the hTRF1 DNA-binding domain in complex with human telomeric DNA was constructed. In addition to DNA recognition by the HTH variant, a flexible N-terminal arm of hTRF1 is likely to interact with DNA
Analysis of Artifacts Caused by Pulse Imperfections in CPMG Pulse Trains in NMR Relaxation Dispersion Experiments
Nuclear magnetic resonance relaxation dispersion (rd) experiments provide kinetics and thermodynamics information of molecules undergoing conformational exchange. Rd experiments often use a Carr-Purcell-Meiboom-Gill (CPMG) pulse train equally separated by a spin-state selective inversion element (U-element). Even with measurement parameters carefully set, however, parts of 1H–15N correlations sometimes exhibit large artifacts that may hamper the subsequent analyses. We analyzed such artifacts with a combination of NMR measurements and simulation. We found that particularly the lowest CPMG frequency (νcpmg) can also introduce large artifacts into amide 1H–15N and aromatic 1H–13C correlations whose 15N/13C resonances are very close to the carrier frequencies. The simulation showed that the off-resonance effects and miscalibration of the CPMG π pulses generate artifact maxima at resonance offsets of even and odd multiples of νcpmg, respectively. We demonstrate that a method once introduced into the rd experiments for molecules having residual dipolar coupling significantly reduces artifacts. In the method the 15N/13C π pulse phase in the U-element is chosen between x and y. We show that the correctly adjusted sequence is tolerant to miscalibration of the CPMG π pulse power as large as ±10% for most amide 15N and aromatic 13C resonances of proteins
NMR Structure of the hRap1 Myb Motif Reveals a Canonical Three-Helix Bundle Lacking the Positive Surface Charge Typical of Myb DNA-Binding Domains
Mammalian telomeres are composed of long tandem arrays of double-stranded telomeric TTAGGG repeats associated with the telomeric DNA-binding proteins, TRF1 and TRF2. TRF1 and TRF2 contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In the budding yeast, telomeric DNA is associated with scRaplp, which has a central DNA-binding domain that contains two structurally related Myb domains connected by a long linker, an N-terminal BRCT domain, and a C-terminal RCT domain. Recently, the human ortholog of scRap1p (hRap1) was identified and shown to contain a BRCT domain and an RCT domain similar to scRap1p. However, hRap1 contained only one recognizable Myb motif in the center of the protein. Furthermore, while scRap1p binds telomeric DNA directly, hRap1 has no DNA-binding ability. Instead, hRap1 is tethered to telomeres by TRF2. Here, we have determined the solution structure of the Myb domain of hRap1 by NMR. It contains three helices maintained by a hydrophobic core. The architecture of the hRap1 Myb domain is very close to that of each of the Myb domains from TRF1, scRap1p and c-Myb. However, the electrostatic potential surface of the hRap1 Myb domain is distinguished from that of the other Myb domains. Each of the minimal DNA-binding domains, containing one Myb domain in TRF1 and two Myb domains in scRap1p and c-Myb, exhibits a positively charged broad surface that contacts closely the negatively charged backbone of DNA. By contrast, the hRap1 Myb domain shows no distinct positive surface, explaining its lack of DNA-binding activity. The hRap1 Myb domain may be a member of a second class of Myb motifs that lacks DNA-binding activity but may interact instead with other proteins. Other possible members of this class are the c-Myb R1 Myb domain and the Myb domains of ADA2 and Adf1. Thus, while the folds of all Myb domains resemble each other closely, the function of each Myb domain depends on the amino acid residues that are located on the surface of each protein. © 2001 Academic Press
Conclusive Evidence of the Reconstituted Hexasome Proven by Native Mass Spectrometry
It has been suggested that the hexasome,
in which one of the H2A/H2B
dimers is depleted from the canonical nucleosome core particle (NCP),
is an essential intermediate during NCP assembly and disassembly,
but little structural evidence of this exists. In this study, reconstituted
products in a conventional NCP preparation were analyzed by native
electrospray ionization mass spectrometry, and it was found that the
hexasome, which migrated in a manner almost identical to that of the
octasome NCP in native polyacrylamide gel electrophoresis, was produced
simultaneously with the octasome NCP. This result might contribute
to understanding the assembly and disassembly mechanism of NCPs
Mass Spectrometric Approach for Characterizing the Disordered Tail Regions of the Histone H2A/H2B Dimer
The
histone H2A/H2B dimer is a component of nucleosome core particles
(NCPs). The structure of the dimer at the atomic level has not yet
been revealed. A possible reason for this is that the dimer has three
intrinsically disordered tail regions: the N- and C-termini of H2A
and the N-terminus of H2B. To investigate the role of the tail regions
of the H2A/H2B dimer structure, we characterized behaviors of the
H2A/H2B mutant dimers, in which these functionally important disordered
regions were depleted, using mass spectrometry (MS). After verifying
that the acetylation of Lys residues in the tail regions had little
effect on the gas-phase conformations of the wild-type dimer, we prepared
two histone H2A/H2B dimer mutants: an H2A/H2B dimer depleted of both
N-termini (dN-H2A/dN-H2B) and a dimer with the N- and C-termini of
H2A and the N-terminus of H2B depleted (dNC-H2A/dN-H2B). We analyzed
these mutants using ion mobility-mass spectrometry (IM-MS) and hydrogen/deuterium
exchange mass spectrometry (HDX-MS). With IM-MS, reduced structural
diversity was observed for each of the tail-truncated H2A/H2B mutants.
In addition, global HDX-MS proved that the dimer mutant dNC-H2A/dN-H2B
was susceptible to deuteration, suggesting that its structure in solution
was somewhat loosened. A partial relaxation of the mutant’s
structure was demonstrated also by IM-MS. In this study, we characterized
the relationship between the tail lengths and the conformations of
the H2A/H2B dimer in solution and gas phases, and demonstrated, using
mass spectrometry, that disordered tail regions play an important
role in stabilizing the conformation of the core region of the dimer
in both phases
Structural Diversity of Nucleosomes Characterized by Native Mass Spectrometry
Histone tails, which
protrude from nucleosome core particles (NCPs),
play crucial roles in the regulation of DNA transcription, replication,
and repair. In this study, structural diversity of nucleosomes was
investigated in detail by analyzing the observed charge states of
nucleosomes reconstituted with various lengths of DNA using positive-mode
electrospray ionization mass spectrometry (ESI-MS) and molecular dynamics
(MD) simulation. Here, we show that canonical NCPs, having 147 bp
DNA closely wrapped around a histone octamer, can be classified into
three groups by charge state, with the least-charged group being more
populated than the highly charged and intermediate groups. Ions with
low charge showed small collision cross sections (CCSs), suggesting
that the histone tails are generally compact in the gas phase, whereas
the minor populations with higher charges appeared to have more loosened
structure. Overlapping dinucleosomes, which contain 14 histone proteins
closely packed with 250 bp DNA, showed similar characteristics. In
contrast, mononucleosomes reconstituted with a histone octamer and
longer DNA (≥250 bp), which have DNA regions uninvolved in
the core-structure formation, showed only low-charge ions. This was
also true for dinucleosomes with free DNA regions. These results suggest
that free DNA regions affect the nucleosome structures. To investigate
the possible structures of NCP observed in ESI-MS, computational structural
calculations in solution and in vacuo were performed. They suggested
that conformers with large CCS values have slightly loosened structure
with extended tail regions, which might relate to the biological function
of histone tails
Gas-Phase Structure of the Histone Multimers Characterized by Ion Mobility Mass Spectrometry and Molecular Dynamics Simulation
The minimum structural unit of chromatin
is the nucleosome core
particle (NCP), consisting of 146 bp of DNA wrapped around a histone
octamer, which itself contains two H2A/H2B dimers and one (H3/H4)<sub>2</sub> tetramer. These multimers possess functionally important
tail regions that are intrinsically disordered. In order to elucidate
the mechanisms behind NCP assembly and disassembly processes, which
are highly related to gene expression, structural characterization
of the H2A/H2B dimer and (H3/H4)<sub>2</sub> tetramer will be of importance.
In the present study, human histone multimers with disordered tail
regions were characterized by electrospray ionization (ESI) ion mobility-mass
spectrometry (IM-MS) and molecular dynamics (MD) simulation. Experimentally
obtained arrival times of these histone multimer ions showed rather
wide distributions, implying that multiple conformers exist for each
histone multimer in the gas phase. To examine their structures, MD
simulations of the histone multimers were performed first in solution
and then <i>in vacuo</i> at four temperatures, resulting
in a variety of histone multimer structures. Theoretical collision
cross-section (CCS) values calculated for the simulated structures
revealed that structural models with smaller CCS values had more compact
tail regions than those with larger CCS values. This implied that
variation of the CCS values of the histone multimers were primarily
due to the random behaviors of the tail regions in the gas phase.
The combination of IM-MS and MD simulation enabled clear and comprehensive
characterization of the gas-phase structures of histone multimers
containing disordered tails