The C-Terminal Zinc Fingers of ZBTB38 are Novel Selective Readers of DNA Methylation.

Pozner A, Hudson NO, Trewhella J, Terooatea TW, Miller SA, Buck-Koehntop BA, J Mol Biol 430(3):258-271 (2018) PubMed

SASDCA3 – Zinc finger and BTB domain-containing protein 38 (ZBTB38) complexed with methylated C-terminal ZBTB38 binding sequence

Zinc finger and BTB domain-containing protein 38
methylated C-terminal ZBTB38 binding sequence
MWI(0) 46 kDa
MWexpected 34 kDa
VPorod 50 nm3
log I(s) 1.80×10-1 1.80×10-2 1.80×10-3 1.80×10-4
Zinc finger and BTB domain-containing protein 38 methylated C-terminal ZBTB38 binding sequence small angle scattering data  s, nm-1
ln I(s)
Zinc finger and BTB domain-containing protein 38 methylated C-terminal ZBTB38 binding sequence Guinier plot ln 1.80×10-1 Rg: 2.5 nm 0 (2.5 nm)-2 s2
(sRg)2I(s)/I(0)
Zinc finger and BTB domain-containing protein 38 methylated C-terminal ZBTB38 binding sequence Kratky plot 1.104 0 3 sRg
p(r)
Zinc finger and BTB domain-containing protein 38 methylated C-terminal ZBTB38 binding sequence pair distance distribution function Rg: 2.6 nm 0 Dmax: 8.2 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Zinc finger and BTB domain-containing protein 38 methylated C-terminal ZBTB38 binding sequence MONSA model

log I(s)
 s, nm-1

log I(s)
 s, nm-1
Zinc finger and BTB domain-containing protein 38 methylated C-terminal ZBTB38 binding sequence HADDOCK model

SAXS data were acquired from solutions of a 1:1 complex between the C-terminal zinc fingers (6-10) of the zinc finger and BTB domain-containing protein 38 (ZBTB38(1006-1153)) and the methylated C-terminal ZBTB38 binding sequence (mCZ38BS) in 10 mM Tris (pH 6.8), 1 mM tris(2-carboxy-ethyl)phosphine (TCEP), 0.05% NaN3 and10% D2O. An in-house Anton Paar SAXSess equipped with a mythen microstrip detector and set with a 10 mm line focus on sample enabled data to be taken immediately after assessment by NMR, which was crucial for characterizing the system within the lifetime of the sample. The data were desmeared by multiplication point by point by the ratio of the desmeared to smeared model P(r) profiles calculated using GNOM with a parameterised slit correction (AH = 2.9 nm-1, LH = 1.5 nm-1) and checked for accuracy against the Anton Paar SAXSQuant desmearing routine based on the Lake algorithm. Scattering data are on an absolute scale (cm-1) set by reference to the scattering from pure H2O. The concentration of the complex was estimated at 3.4 mg/mL, but the error is large as the complex was prepared using a concentration step after combining components and concentration estimated by change in volume which results in a significant error in the molecular mass determination from I(0) (30%). However, the Porod volume from the scattering data indicates the molecular mass of the scattering particle is within 3% of that expected for a 1:1 stoichiometry ZBTB38(1006-1153):mCZ38BS. MULCh was used to calculate the average partial specific volume (0.657 mL/g) and X-ray contrast (Δρ = 4.12e10 cm-2). Two phase (protein and DNA) modelling using MONSA, with simultaneous fitting of the ZBTB38(1006-1153):mCZ38BS complex and free mCZ38BS data, show the protein binds at one end of the duplex DNA. To generate an atomistic model, a homology model for the five C-terminal ZBTB38 ZFs was built within SWISS-MODEL using ZFs 2-6 from the protein Aart in complex with DNA (PDB: 2I13) as the template. A model for CZ38BS was built in w3DNA after which the 3D-DART web server was utilized to introduce a widening of the major groove at the core ATCGGCG recognition motif and to introduce a 20° bend between residues 12-27. Docking of the ZBTB38 ZFs onto the CZ38BS was performed using the HADDOCK web server (version 2.2). The position of ZF10 (residues 1125-1153) in this model was uncertain, and so two sets of rigid body calculations were performed against the SAXS data; one with no protein:DNA constraints specified, and one including weak 10 Å distance constraints that maintained ZF10 in position to interact at the end of the DNA (between the phosphates of G1-A3 and the Cα of Lys1146, and the phosphates of G55-G57 and the Cα of Lys1145). From these calculations, there was no real consensus on the exact positioning of ZF10 and ensemble modelling did not show any improvement in the fits. The atomistic model deposited here with its CRYSOL fit was obtained from SASREF including the protein-DNA contact constraints involving ZF10 which is a good fit to the SAXS data, agrees reasonably with the MONSA model, and is presented with the understanding that it represents a hybrid model that fits the currently available data and the positioning of ZF10 remains uncertain.

Zinc finger and BTB domain-containing protein 38 (ZBTB38)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   17.3 kDa
 
UniProt   Q8NAP3 (1006-1152)
Sequence   FASTA
 
methylated C-terminal ZBTB38 binding sequence (mZBTB38BS)
Mol. type   DNA
Olig. state   Monomer
Mon. MW   16.7 kDa
Sequence   FASTA