Interactions of protein side chains with RNA defined with REDOR solid state NMR

Abstract

Formation of the complex between human immunodeficiency virus type-1 Tat protein and the transactivation response region (TAR) RNA is vital for transcriptional elongation, yet the structure of the Tat-TAR complex remains to be established. The NMR structures of free TAR, and TAR bound to Tat-derived peptides have been obtained by solution NMR, but only a small number of intermolecular NOEs could be identified unambiguously, preventing the determination of a complete structure. Here we show that a combination of multiple solid state NMR REDOR experiments can be used to obtain multiple distance constraints from (15)N to (13)C spins within the backbone and side chain guanidinium groups of arginine in a Tat-derived peptide, using (19)F spins incorporated into the base of U23 in TAR and (31)P spins in the P22 and P23 phosphate groups. Distances between the side chain of Arg52 and the base and phosphodiester backbone near U23 measured by REDOR NMR are comparable to distances observed in solution NMR-derived structural models, indicating that interactions of TAR RNA with key amino acid side chains in Tat are the same in the amorphous solid state as in solution. This method is generally applicable to other protein-RNA complexes where crystallization or solution NMR has failed to provide high resolution structural information.

Nucleotide Binding Modes in a Motor Protein Revealed by 31P‐ and 1H‐Detected MAS Solid‐State NMR Spectroscopy

“…The sensitivity of 1 Hc hemicalshift values on noncovalent interactions involved in these molecular recognition processes is exploiteda llowing us to probe directly the chemical bonding state, an information, which is not directly accessible from an X-ray structure. [9][10][11][12] Twoa pproaches are particularly promising to probe nucleotide interactions:p hosphorus-( 31 P) and proton-( 1 H) detected spectroscopy.D istances between 31 Ps pins of DNA and 15 N spins of ap rotein have been measuredb yu sing transferredecho, double-resonance (TEDOR) experiments. Binding of nucleotides, such as ATPa nd DNA, occurs throughn oncovalent interactions includingh ydrogen bonds, electrostatic (salt bridges), and van der Waals interactions [3,4] (the latter also comprising interactions between aromatic rings [5] ).…”

“…[4,6,7] Still, many of the scenarios described above involve protein complexes, which are difficult to crystallize, and when they do so, might reflect at insufficient resolution to clearly identify interactions.A lternative methods are therefore neededa nd can be providedt hrough solid-state NMR spectroscopy,w hich can access also large biomolecular complexes,a nd importantly in sample formats where the assemblies are simply sedimented into the NMR rotor. [9][10][11][12] Twoa pproaches are particularly promising to probe nucleotide interactions:p hosphorus-( 31 P) and proton-( 1 H) detected spectroscopy.D istances between 31 Ps pins of DNA and 15 N spins of ap rotein have been measuredb yu sing transferredecho, double-resonance (TEDOR) experiments. [9][10][11][12] Twoa pproaches are particularly promising to probe nucleotide interactions:p hosphorus-( 31 P) and proton-( 1 H) detected spectroscopy.D istances between 31 Ps pins of DNA and 15 N spins of ap rotein have been measuredb yu sing transferredecho, double-resonance (TEDOR) experiments.…”

Preparation of RNA samples with narrow line widths for solid state NMR investigations

“…Thus, characterization of the structure and dynamics of TAR RNA is critical to understand the interactions between RNA and Tat protein. Therefore, the structures of TAR have been widely studied [9,10,[15][16][17][18][19], but a structure of the complex is still missing. Furthermore, characterization of the dynamics of TAR remains incomplete because motions occur on multiple time scales and not all are accessible to solution NMR studies.…”

A Suite of Solid‐State NMR Experiments for RNA Intranucleotide Resonance Assignment in a 21 kDa Protein–RNA Complex

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Solid-state NMR (ssNMR) spectroscopy is becoming a very important instrument for the elucidation of structure-function relationships in large biomolecular complexes. To date, substantial progresses have been made in the structure determination of membrane proteins [1][2][3] and amyloid fibrils, [4][5][6][7] whereas significantly fewer studies have addressed the structure of RNA [8][9][10][11][12][13][14][15][16][17] or protein-RNA complexes (RNP) [13][14][15][16]18] by ssNMR spectroscopy. Nevertheless, the application of ssNMR spectroscopy to study large RNP complexes holds excellent promises, because of the independence of the ssNMR line widths from the molecular size.Here, we present a comprehensive ssNMR-based intranucleotide resonance assignment of the 26mer box C/D RNA in complex with L7Ae (21 kDa in molecular weight, Figure 1 a).

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“…In a first attempt, we prepared the L7Ae-box C/D RNA complex using uniformly labeled 13 C, 15 N box C/D RNA (u-RNA). The resolution obtained in the 13 C CP (cross polarization) experiment, as well as in the 13 C, 13 C PDSD experiment, was poor because of strong resonance overlap (Figure S1a), an observation that revealed the need for nucleotide-type selective labeling.…”