PDBsum entry 2d3d

RNA binding protein

PDB id: 2d3d

Contents

Protein chain

83 a.a. *

Metals

_CA

Waters ×73

* Residue conservation analysis

PDB id:

2d3d

Name:

RNA binding protein

Title:

Crystal structure of the RNA binding sam domain of saccharomyces cerevisiae vts1

Structure:

Vts1 protein. Chain: a. Fragment: minimal RNA binding fragment. Engineered: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: vts1. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

1.60Å R-factor: 0.208 R-free: 0.256

Authors:

T.Aviv,A.N.Amborski,X.S.Zhao,J.J.Kwan,P.E.Johnson,F.Sicheri, L.W.Donaldson

Key ref:

T.Aviv et al. (2006). The NMR and X-ray structures of the Saccharomyces cerevisiae Vts1 SAM domain define a surface for the recognition of RNA hairpins. J Mol Biol, 356, 274-279. PubMed id: 16375924 DOI: 10.1016/j.jmb.2005.11.066

Date:

27-Sep-05 Release date: 14-Feb-06

Protein chain

Q08831  (VTS1_YEAST) -  RNA-binding protein VTS1 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 523 a.a.

Struc: 83 a.a.

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1016/j.jmb.2005.11.066

J Mol Biol 356:274-279 (2006)

PubMed id: 16375924

The NMR and X-ray structures of the Saccharomyces cerevisiae Vts1 SAM domain define a surface for the recognition of RNA hairpins.

T.Aviv, A.N.Amborski, X.S.Zhao, J.J.Kwan, P.E.Johnson, F.Sicheri, L.W.Donaldson.

ABSTRACT

The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator Vts1 has a high affinity towards RNA hairpins containing a CUGGC pentaloop. We present the 1.6 Angstroms X-ray crystal structure of the Vts1 SAM domain in its unliganded state, and the NMR solution structure of this domain in its RNA-bound state. Both structures reveal a canonical five helix SAM domain flanked by additional secondary structural elements at the N and C termini. The two structures are essentially identical, implying that no major structural rearrangements occur upon RNA binding. Amide chemical shift changes map the RNA-binding site to a shallow, basic patch at the junction of helix alpha5 and the loop connecting helices alpha1 and alpha2.

Selected figure(s)

Figure 1.
Figure 1. The crystal structure of free Vts1 SAM and the NMR structure of SRE-bound Vts1 SAM are essentially identical. A gene fragment encompassing the SAM domain (residues 407-523) of S. cerevisiae VTS1 was amplified by PCR methods and inserted into a modified pGEX vector. The resulting glutathione-S-transferase fusion protein was purified by affinity chromatography from soluble extracts of E. coli BL21(DE3) grown in a BioFlow 110 fermentor at 25 °C. Isotopically labeled Vts1^407-526 was obtained from fermentations in minimal M9 medium containing 1.0 g/l [U-98%] [15N]ammonium chloride and 4.0 g/l [U-99%] [13C]glucose as the sole sources of nitrogen and carbon, respectively. Vts1^407-523 was cleaved from the fusion protein with TEV protease. Proteins were further purified and buffer-exchanged using S-100 gel-filtration chromatography. For crystallography, the 13 kDa Vts1 fragment was digested with thermolysin to produce a minimal 10 kDa fragment, Vts1^436-523, that was purified further by heparin chromatography. A 19 nt SRE RNA (5'-GGAGGCUCUGGCAGCUUUC-3') was prepared in milligram quantities for NMR spectroscopy by phage T7 RNA polymerase-driven, in vitro transcription.17 The SRE RNA was purified by denaturing 20% PAGE and electroeluted. Renaturation of the SRE RNA hairpin was achieved by rapidly chilling a solution that was preheated to 94 °C. (a) A fluorescence polarization binding assay7 of the thermolysin-resistant Vts1 SAM domain demonstrates high-affinity binding for the SRE (5'-AGGCUCUGGCAGUCU-3') but not a mutant SRE bearing substitutions at the first and third loop positions. (b) A modest decrease in binding affinity is observed when either 5 mM EDTA or 10 mM Ca^2+ is present in the binding buffer. (c) Superposition of the free Vts1 SAM domain crystal structure with the bound NMR structure reveals no significant conformational changes. The SRE binding site is indicated in red. Superposition of the crystal structures of the Vts1 SAM domain with the (d) EphA4 SAM domain and the (e) Smaug SAM domain. (f) Superposition of the 20 lowest energy NMR structures of the RNA-bound Vts1 SAM domain.

Figure 2.
Figure 2. The RNA-binding surface on the Vts1 SAM domain. (a) Weighted-averaged18 amide 1H and 15N chemical shift changes upon SRE RNA binding where d[av](HN)=[dH2+(dN/5)2)/2]1/2. Bars are colored according to the magnitude of the chemical shift change (red, >0.3 ppm; yellow, >0.1 ppm). (b) Chemical shift changes, sequence conservation, and electrostatic charge are mapped onto the surface of the free X-ray structure of Vts1 SAM domain. HSQC perturbations are colored according to (a). The linewidth of Lys467 sharpens significantly upon RNA binding and is colored orange. Sequence identity (in purple) and sequence similarity (in pink) among Vts1 homologs coincides with the SRE binding surface deduced from chemical shift perturbations. The RNA-binding site demonstrates basic charge distribution (red; negative potential; blue, positive potential). Molecular structure representations and electrostatic calculations were made with pyMOL (http://pymol.sourceforge.net).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 356, 274-279) copyright 2006.