Determination of the Secondary Structure and Folding Topology of an RNA Binding Domain of Mammalian hnRNP A1 Protein Using Three-Dimensional Heteronuclear Magnetic Resonance Spectroscopy

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States
Biochemistry (Impact Factor: 3.02). 04/1994; 33(10):2852-8. DOI: 10.1021/bi00176a015
Source: PubMed


The secondary structure and folding topology of the first RNA binding domain of the human hnRNP A1 protein was determined by multidimensional heteronuclear NMR spectroscopy. The 92 amino acid long domain exhibits a beta alpha beta beta alpha beta folding pattern, arranged in a four-stranded antiparallel beta-sheet flanked by two alpha-helices, which is very similar to that found for other members of this family. Regions of marked variation between the structurally characterized RNA binding proteins of this class to date are mainly localized in the loops connecting the secondary structure elements.

4 Reads
  • Source
    • "The HNCO experiment correlates the amide HN and 15N chemical shift of one amino acid with the carbonyl (C’) shift of the preceding residue by using the one-bond 15N-13CO J-coupling to establish the sequential correlation. As shown in Figure 4d, HN of residue of T41 is connected to C’ of V40, in the 13C(F1)-1H(F3) plane at δ[15N(F3)] = 116.4 ppm of the 3D HNCO of RGD hirudin (1–66) From the combination of CBCA(CO)NH and CBCANH experiments, the backbone resonance assignments and the sequential connectivities can be obtained, which can help to resolve a number of uncertainties in assignments due to degeneracy or the close similarity of backbone chemical shifts [24]. Almost complete 15N, 13 C and 1H resonance assignments of the backbone in r-RGD-Hirudin are reported (Table 3). "
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.
    PLoS ONE 08/2012; 7(8):e42207. DOI:10.1371/journal.pone.0042207 · 3.23 Impact Factor
  • Source
    • "90% of the polypeptide. The polypeptide corresponding to the N-terminal half of UP1 was analyzed and the assignments were reported (36). It was confirmed that our assignments for the whole UP1 are consistent with those reported for the N-terminal half. "
    [Show abstract] [Hide abstract]
    ABSTRACT: We found that UP1, a proteolytic product of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), both enhances and represses the telomerase activity. The formation of the UP1-telomerase RNA-telomeric DNA ternary complex was revealed by a gel retardation experiment. The interactions in the ternary and binary complexes were elucidated by NMR. UP1 has two nucleic acid-binding domains, BD1 and BD2. In the UP1-telomerase RNA binary complex, both BD1 and BD2 interact with telomerase RNA. Interestingly, when telomeric DNA was added to the binary complex, telomeric DNA bound to BD1 in place of telomerase RNA. Thus, BD1 basically binds to telomeric DNA, while BD2 mainly binds to telomerase RNA, which resulted in the formation of the ternary complex. Here, UP1 bridges telomerase and telomeric DNA. It is supposed that UP1/hnRNP A1 serves to recruit telomerase to telomeric DNA through the formation of the ternary complex. A model has been proposed for how hnRNP A1/UP1 contributes to enhancement of the telomerase activity through recruitment and unfolding of the quadruplex of telomeric DNA.
    Nucleic Acids Research 11/2008; 36(21):6816-24. DOI:10.1093/nar/gkn767 · 9.11 Impact Factor
  • Source
    • "More importantly , secondary structure predictions using the neural network method PHD (Rost et al., 1994) and the hydrophobic cluster analysis (HCA; reviewed in Callebaut et al., 1997) reveal a secondary structure for domain B composed of four ␤ strands and one ␣-helix in the same arrangement as classical RNP folding, with the putative RNP-2 and RNP-1 located on ␤1 and ␤3, respectively, as expected. This is shown in Fig. 6 where domain B is compared to the RNP domain of hnRNP A1 whose secondary structure prediction has been confirmed by physicochemical methods (Garrett et al., 1994). Concerning helix ␣2, the PHD method predicts at this position a ␤ strand with a moderate score, whereas the HCA method predicts an ␣-helix which is more in agreement with the structure of demonstrated RNP domains. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Potyvirus helper component-proteinase (HC-Pro) binds nonspecifically to single-stranded nucleic acids with a preference for RNA. To delineate the regions of the protein responsible for RNA binding, deletions were introduced into the full-length Potato potyvirus Y HC-Pro gene carried by an Escherichia coli expression vector. The corresponding proteins were expressed as fusions with the maltose-binding protein, purified, and assayed for their RNA-binding capacity. The results obtained by UV cross-linking and Northwestern blot assays demonstrated that the N- and C-terminal regions of HC-Pro are dispensable for RNA binding. They also revealed the presence of two independent RNA-binding domains (designated A and B) located in the central part of HC-Pro. Domain B appears to contain a ribonucleoprotein (RNP) motif typical of a large family of RNA-binding proteins involved in several cellular processes. The possibility that domain B consists of an RNP domain is discussed and suggests that HC-Pro could constitute the first example of a plant viral protein belonging to the RNP-containing family of proteins.
    Virology 04/2000; 268(1):104-11. DOI:10.1006/viro.1999.0156 · 3.32 Impact Factor
Show more