pubmed.ncbi.nlm.nih.gov

Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling - PubMed

  • ️Sun Jan 01 2006

Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling

Francesca Vitali et al. EMBO J. 2006.

Abstract

The polypyrimidine tract binding protein (PTB) is a 58 kDa protein involved in many aspects of RNA metabolism. In this study, we focused our attention on the structure of the two C-terminal RNA recognition motifs (RRM3 and RRM4) of PTB. In a previous study, it was found that the two RRMs are independent in the free state. We recently determined the structure of the same fragment in complex with RNA and found that the two RRMs interact extensively. This difference made us re-evaluate in detail the free protein structure and in particular the interdomain interface. We used a combination of NMR spectroscopy and segmental isotopic labeling to unambiguously study and characterize the interdomain interactions. An improved segmental isotopic labeling protocol was used, enabling us to unambiguously identify 130 interdomain NOEs between the two RRMs and to calculate a very precise structure. The structure reveals a large interdomain interface, resulting in a very unusual positioning of the two RRM domains relative to one another.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Protein constructs used in this study. Protein domains are represented by colored boxes: orange for the NLS sequence; white for linker regions; pink for PTB RRM1; violet for PTB RRM2; red for PTB RRM3 domain; yellow for PTB RRM4; gray for 6 × His tag; blue for CBD; light blue for the Mxe GyrA intein; and green for the Ssp DnaB intein.

Figure 2
Figure 2

NMR spectra of full-length PTB and several PTB subfragments. (A) 15N-TROSY of full–length PTB recorded at 900 MHz, 303 K, pH 5.8. (B) Overlay of 15N-TROSY of PTB RRM1 (red), RRM2 (green) and RRM34 (blue) recorded under the same condition as in panel A. (C) 15N-HSQC of PTB RRM34 recorded at 500 MHz, 303 K, pH 6.5. (D) Overlay of 15N-HSQC recorded at 500 MHz, 303 K, pH 6.5 of PTB RRM3 (blue) and RRM4 (green).

Figure 3
Figure 3

Segmental labeling of PTB RRM34. (A) Intein-mediated EPL. (i) N → S acyl shift of Cys at the intein N-terminus resulting in the formation of a reactive thioester. (ii) Thiol-mediated cleavage: a nucleophilic attack on the thioester by a small thiol compound (MESNA) cleaves the precursor protein and generates a new thioester at the C-terminus of the target protein. (iii) Cyclization of the Ssp DnaB intein C-terminal Asn releases the α-Cys domain from the succinamide derivative of the intein. (iv) The ligation reaction utilizes identical coupling chemistry as the native chemical ligation, and the activated thioester is attacked by the α-Cys residue. (v) An S → N acyl shift, a spontaneous rearrangement of the thioester, results in the formation of a peptide bond between the two domains. (B) SDS–PAGE of PTB RRM34 and subdomains. Lane 1: protein marker; lane 2: PTB RRM3 after chitin affinity column purification; lane 3: PTB RRM4 after chitin affinity column purification; lane 4: mixture of a PTB RRM34 ligation reaction following a standard EPL protocol (Severinov and Muir, 1998; Xu et al, 1999); lane 5: mixture of a PTB RRM34 ligation reaction performed on column; lane 6: PTB RRM34 ligated on column after the Ni-NTA purification. (C) Electrospray mass spectra of purified ligated PTB RRM34.

Figure 4
Figure 4

NMR spectra of the segmentally labeled PTB RRM34. (A) 15N-HSQC of RRM34 (S443C) with 15N,13C-labeled RRM3 ligated to 15N-only-labeled RRM4, recorded at 900 MHz, 303 K, pH 6.5. (B) Overlay of two 15N-HSQC of PTB RRM34 (S443C) segmentally labeled, under the same condition as panel A. 15N,13C–labeled RRM3 is indicated by blue peaks and 15N,13C–labeled RRM4 by green peaks. (C, D) Cross-sections of a 3D 13C-edited half-filter NOESY of the segmentally labeled RRM34 where RRM3 is 13C-labeled. (C) Several interdomain NOEs to I356 γ2 can be observed in the 13C plane at 18.6 ppm. (D) Several interdomain NOEs to V360 γ1 can be observed in the 13C plane at 24.8 ppm. (E) Cross-section of a 3D 13C-edited half-filter NOESY of the segmentally labeled RRM34 where RRM4 is 13C-labeled. Several interdomain NOEs to I449 and I509 γ2 can be observed in the 13C plane at 18.6 ppm.

Figure 5
Figure 5

Structure of free and bound PTB RRM34. (A) Superimposition of the 20 lowest energy conformers of RRM34 in its free form. Protein side chains contributing to the interdomain interaction are displayed in blue (residues 324–442 that include RRM3) and in green (residues 443–531 that include RRM4). (B) Stereo view of the lowest energy conformer of PTB RRM34. (C) Stereo view of PTB RRM34 in complex with RNA (Oberstrass et al, 2005). The RNA is shown in yellow.

Figure 6
Figure 6

Close-up view of the interdomain interface in PTB RRM34 and hnRNPA1 RRM12. (A) Stereo view of the interaction between helix 2 of RRM4 and helix 1 of RRM3. Side chains for RRM3 and RRM4 and the interdomain linker are represented by sticks and dotted surfaces colored in blue, green and red, respectively. (B) Stereo view of the interaction between helix 2 of RRM3 with the interdomain linker and F526 from RRM4. (C) Stereo view of the interdomain interface found in the structure of hnRNPA1 RRM12 (Shamoo et al, 1997).

Figure 7
Figure 7

Relaxation measurement and mutagenesis of PTB RRM34. (A) 15N–1H NOE values of the backbone amide resonances of PTB RRM34 plotted against the residue number. (B) T1 values of the backbone amides. (C) T2 values of the backbone amides. (D) 15N-HSQC spectra of PTB RRM34 containing six side-chain mutations (I356K, F446E, I449K, E502K, V505E and I509K) at the interdomain interface.

Similar articles

Cited by

References

    1. Allain FH, Bouvet P, Dieckmann T, Feigon J (2000a) Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin. EMBO J 19: 6870–6881 - PMC - PubMed
    1. Allain FH, Gilbert DE, Bouvet P, Feigon J (2000b) Solution structure of the two N-terminal RNA-binding domains of nucleolin and NMR study of the interaction with its RNA target. J Mol Biol 303: 227–241 - PubMed
    1. Bax A, Clore GM, Gronenborn AM (1990) 1H–1H correlation via isotropic mixing of 13C magnetization, a new three-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J Magn Reson 88: 425–431
    1. Beligere GS, Dawson PE (1999) Conformationally assisted protein ligation using C-terminal thioester peptides. J Am Chem Soc 121: 6332–6333
    1. Camarero JA, Shekhtman A, Campbell EA, Chlenov M, Gruber TM, Bryant DA, Darst SA, Cowburn D, Muir TW (2002) Autoregulation of a bacterial σ factor explored by using segmental isotopic labeling and NMR. Proc Natl Acad Sci USA 99: 8536–8541 - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources