Abstract 3093: 3D structural report of NRAS isoform 5
Authors: Markowitz Joseph , Mal Tapas K. , Yuan Chunhua , Courtney Nicholas B. , Patel Mitra , Stiff Andrew R. , Blachly James , Walker Christopher , Eisfeld Ann-Kathrin , de la Chapelle Albert , Carson William E. 
Published: July 2016

The protein NRAS is part of the NRAS-BRAF-MEK-ERK signaling cascade. Recently, 5 NRAS isoforms were discovered by our group, and it was observed that isoform 5 overexpression in vitro resulted in a more aggressive cell phenotype. NRAS isoform 5 increases phosphorylation of downstream targets (AKT, MEK, and ERK). The structural report is unavailable for NRAS isoform 5. In addition, it was found in the current study that the NRAS isoform 5 does not have GTPase activity due to lack the canonical GTP binding region. Given the importance of this pathway in driving melanoma progression, we studied the structural details of this novel NRAS isoform 5 by two biophysical techniques, NMR and CD spectroscopy.

The circular dichroism spectra of NRAS isoform 5 that was C-acetylated and N-amidated was measured using 11%, 14%, 21%, 45%, 56% and 85% trifluoroethanol (TFE) in a phosphate buffer. The secondary structural elements were induced at ∼14-15% TFE. There were no qualitative differences in the CD spectra between 56% and 85% TFE. Therefore 56% deuterated TFE was utilized in the NMR structural studies.

Two dimensional (2D) homonuclear and heteronuclear experiments were acquired at 25°C with Bruker Avance III HD 600 and 800 MHz NMR spectrometers (Campus Chemical Instrument Center NMR facility), each equipped with a triple resonance z axis gradient TXI cryoprobe for amino acid chemical shift assignments and structural determination of NRAS isoform 5. The NMR chemical shifts for the protons were assigned using traditional 2D TOCSY, COSY, and NOESY experiments. The 2D proton-carbon HSQC was utilized to facilitate the assignment of the alpha and side chain aliphatic protons. In the absence of TFE, there were no observable NOEs that could represent ordered secondary structures. Other than intra residue and short range sequential NOEs (i-j ≤ 2), only a weak medium range NOE was observed from the aromatic protons of Tyr 4 to the gamma proton of Val 7/8.

The secondary structure of NRAS isoform 5 was determined via the same NMR techniques in 56% TFE. The secondary structure was defined by 69 sequential NOEs and 39 medium range NOEs. NOEs characteristic of helical structure were observed in the NOESY spectra. For example, NOEs were measured from H(alpha, i) to H(beta, i+3) for residues Tyr 4, Lys 5, and Leu 6. The tertiary fold was determined by 10 key long-range NOEs. The flexible C terminal is brought in close proximity to the N-terminal helix by 4 NOEs between Val 8/9 and Val 14, and 5 NOEs between Tyr 4 and Try 20 to form a helix-turn-coil structure in presence of TFE. In addition, the proton chemical shifts for the aromatic proton chemical shifts for the tyrosine aromatic ring became distinct.

NRAS isoform 5 is highly flexible in aqueous solution, but forms a helix-turn-coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy. This data may be utilized as a starting point to understand the biophysical interactions of this novel NRAS isoform.

Citation Format: Joseph Markowitz, Tapas K. Mal, Chunhua Yuan, Nicholas B. Courtney, Mitra Patel, Andrew R. Stiff, James Blachly, Christopher Walker, Ann-Kathrin Eisfeld, Albert de la Chapelle, William E. Carson. 3D structural report of NRAS isoform 5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3093.