24 research outputs found
Hydrogen–deuterium exchange strategy for delineation of contact sites in protein complexes
AbstractWe use NMR spectra to determine protein–protein contact sites by observing differences in amide proton hydrogen–deuterium exchange in the complex compared to the free protein in solution. Aprotic organic solvents are used to preserve H/D labeling patterns that would be scrambled in water solutions. The binding site between the mammalian co-chaperone Aha1 with the middle domain of the chaperone Hsp90 obtained by our H/D exchange method corresponds well with that in the X-ray crystal structure of the homologous complex from yeast, even to the observation of a secondary binding site. This method can potentially provide data for complexes with unknown structure and for large or dynamic complexes inaccessible via NMR and X-ray methods
The CH2 domain of CBP/p300 is a novel zinc finger
AbstractThe transcriptional co-regulator CBP (CREB-binding protein) has a highly conserved cysteine/histidine-rich region (CH2) whose structure and function remain uncharacterized. Using nuclear magnetic resonance (NMR spectroscopy), sequence alignment, mass spectrometry, and mutagenesis, we show that the CH2 domain is not a canonical plant homeodomain (PHD) finger, as previously proposed, but binds an additional zinc atom through the region N-terminal to the putative PHD motif. The CH2 domain and the preceding bromodomain interact and mutually stabilize each other, implying a cooperative function. We tested the hypothesis that the bromodomain and the CH2 domain can interact with histones, but found that the CH2 does not participate in histone-recognition
Solution Structure of the N-terminal Zinc Fingers of the Xenopus laevis double-stranded RNA-binding Protein ZFa
Several zinc finger proteins have been discovered recently that bind specifically to double-stranded RNA. These include the mammalian JAZ and wig proteins, and the seven-zinc finger protein ZFa from Xenopus laevis. We have determined the solution structure of a 127 residue fragment of ZFa, which consists of two zinc finger domains connected by a linker that remains unstructured in the free protein in solution. The first zinc finger consists of a three-stranded beta-sheet and three helices, while the second finger contains only a two-stranded sheet and two helices. The common structures of the core regions of the two fingers are superimposable. Each finger has a highly electropositive surface that maps to a helix-kink-helix motif. There is no evidence for interactions between the two fingers, consistent with the length (24 residues) and unstructured nature of the intervening linker. Comparison with a number of other proteins shows similarities in the topology and arrangement of secondary structure elements with canonical DNA-binding zinc fingers, with protein interaction motifs such as FOG zinc fingers, and with other DNA-binding and RNA-binding proteins that do not contain zinc. However, in none of these cases does the alignment of these structures with the ZFa zinc fingers produce a consistent picture of a plausible RNA-binding interface. We conclude that the ZFa zinc fingers represent a new motif for the binding of double-stranded RNA
Structure of the p53 Transactivation Domain in Complex with the Nuclear Receptor Coactivator Binding Domain of CREB Binding Protein
Mapping the Interactions of the p53 Transactivation Domain with the KIX Domain of CBP â€
Hydrogen-deuterium exchange strategy for delineation of contact sites in protein complexes
Structural Characterization of Interactions between the Double-Stranded RNA-Binding Zinc Finger Protein JAZ and Nucleic Acids
The interactions of the human double-stranded
RNA-binding zinc
finger protein JAZ with RNA or DNA were investigated using electrophoretic
mobility-shift assays, isothermal calorimetry, and nuclear magnetic
resonance spectroscopy. Consistent with previous reports, JAZ has
very low affinity for duplex DNA or single-stranded RNA, but it binds
preferentially to double-stranded RNA (dsRNA) with no detectable sequence
specificity. The affinity of JAZ for dsRNA is unaffected by local
structural features such as loops, overhangs, and bulges, provided
a sufficient length of reasonably well-structured A-form RNA (about
18 bp for a single zinc finger) is present. Full-length JAZ contains
four Cys<sub>2</sub>His<sub>2</sub> zinc fingers (ZF1–4) and
has the highest apparent affinity for dsRNA; two-finger constructs
ZF12 and ZF23 have lower affinity, and ZF34 binds even more weakly.
The fourth zinc finger, ZF4, has no measurable RNA-binding affinity.
Single zinc finger constructs ZF1, ZF2, and ZF3 show evidence for
multiple-site binding on the minimal RNA. Fitting of quantitative
NMR titration and isothermal calorimetry data to a two-site binding
model gave <i>K</i><sub>d1</sub> ∼ 10 μM and <i>K</i><sub>d2</sub> ∼ 100 μM. Models of JAZ–RNA
complexes were generated using the high-ambiguity-driven biomolecular
docking (HADDOCK) program. Single zinc fingers bind to the RNA backbone
without sequence specificity, forming complexes with contacts between
the RNA minor groove and residues in the N-terminal β strands
and between the major groove and residues in the helix–kink–helix
motif. We propose that the non-sequence-specific interaction between
the zinc fingers of JAZ with dsRNA is dependent only on the overall
shape of the A-form RNA