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The small nuclear 7SK RNA regulates RNA polymerase II (RNA Pol II) transcription, by sequestering and inhibiting the positive transcription elongation factor b (P-TEFb). P-TEFb is stored in the 7SK ribonucleoprotein (RNP) that contains the three nuclear proteins Hexim1, LaRP7, and MePCE. P-TEFb interacts with the protein Hexim1 and the 7SK RNA. Once P-TEFb is released from the 7SK RNP, it activates transcription by phosphorylating the C-terminal domain of RNA Pol II. P-TEFb also plays a crucial role in the replication of the human immunodeficiency virus HIV-1, through its recruitment by the viral transactivator Tat. Previous work demonstrated that the protein Tat promotes the release of P-TEFb from the 7SK RNP through direct binding to the 7SK RNA. Hexim1 and Tat proteins both comprise conserved and similar arginine-rich motifs that were identified to bind the 7SK RNA at a repeated GAUC site located at the top of the 5′-terminal hairpin (HPI). Here, we report the solution structure of this region as determined by nuclear magnetic resonance, to identify HPI structural features recognized by Hexim1 and Tat. The HPI solution structure displays an elongated shape featuring four helical segments interrupted by one internal loop and three bulges with distinct folds. In particular, the repeated GAUC motif adopts a pre-organized geometry. Our results suggest that the binding of Hexim1 and Tat to the 7SK RNA could originate from a conformational selection of this motif, highlighting how RNA local structure could lead to an adaptive recognition of their partners.

Interleukin-8alpha (IL-8alpha) is an antimicrobial peptide derived from the chemokine IL-8. Solution NMR was used to determine the atomic-resolution structure of IL-8alpha in SDS micelles. Solid-state NMR and tryptophan fluorescence were used to probe the interaction of IL-8alpha with model membranes. The peptide interacted differently with anionic versus purely zwitterionic micelles or bilayers. Tryptophan fluorescence demonstrated a deeper position of Trp4 in SDS micelles and POPC/POPG bilayers compared to pure POPC bilayers, consistent with (2)H order parameters, which also indicated a deeper position of the peptide in POPC/POPG bilayers compared to POPC bilayers. Paramagnetic probe data showed that IL-8alpha was situated roughly parallel to the SDS micelle surface, with a slight tilt that positioned the N-terminus more deeply in the micelle compared to the C-terminus. (15)N solid-state NMR spectra indicated a similar, nearly parallel position for the peptide in POPC/POPG bilayers. (31)P and (2)H solid-state NMR demonstrated that the peptide did not induce the formation of any nonlamellar phases and did not significantly disrupt bilayer orientation in aligned model membranes composed of POPC or POPC and POPG.

Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies.

As a major component of the innate immune system, natural killer cells are responsible for activating the cytolytic killing of certain pathogen-infected or tumor cells. The self-recognition of natural killer cells is achieved in part by the killer cell immunoglobulin-like receptors (KIRs) protein family. In the current study, using a suite of biophysical methods, we investigate the self-association of an activating KIR, KIR2DS1. This KIR is of particular interest because when in the presence of the HLA-Cw6 protein, KIR2DS1 becomes a major risk factor for psoriasis, an autoimmune chronic skin disease. Using circular dichroism spectroscopy, dynamic light scattering, and atomic force microscopy, we reveal that KIR2DS1 self-associates in a well-defined fashion. Our novel results on an activating KIR allow us to suggest a working model for the KIR2DS1- HLA class I molecular mechanism.

Topologically, platelet factor-4 kinocidins consist of distinct N-terminal extended, C-terminal helical, and interposing gamma-core structural domains. The C-terminal alpha-helices autonomously confer direct microbicidal activity, and the synthetic antimicrobial peptide RP-1 is modeled upon these domains. In this study, the structure of RP-1 was assessed using several complementary techniques. The high-resolution structure of RP-1 was determined by NMR in anionic sodium dodecyl sulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles, which approximate prokaryotic and eukaryotic membranes, respectively. NMR data indicate the peptide assumes an amphipathic alpha-helical backbone conformation in both micelle environments. However, small differences were observed in the side-chain orientations of lysine, tyrosine, and phenylalanine residues in SDS versus DPC environments. NMR experiments with a paramagnetic probe indicated differences in positioning of the peptide within the two micelle types. Molecular dynamics (MD) simulations of the peptide in both micelle types were also performed to add insight into the peptide/micelle interactions and to assess the validity of this technique to predict the structure of peptides in complex with micelles. MD independently predicted RP-1 to interact only peripherally with the DPC micelle, leaving its spherical shape intact. In contrast, RP-1 entered deeply into and significantly distorted the SDS micelle. Overall, the experimental and MD results support a preferential specificity of RP-1 for anionic membranes over zwitterionic membranes. This specificity likely derives from differences in RP-1 interaction with distinct lipid systems, including subtle differences in side chain orientations, rather than gross changes in RP-1 structure in the two lipid environments.

Antimicrobial peptides are naturally occurring molecules, part of the innate immune system, and are of high interest as novel antibiotic therapeutics given the increasing resistance of microbes to conventional antibiotics. RP-1 and IL-8α are 18 and 19 amino acid synthetic peptides that were designed based on the sequence of the C-terminal helical segments of two chemokines: platelet factor-4 and interleukin-8. In order to characterize structure-activity relationships and to understand the selectivity of these peptides for bacterial membranes, NMR was used to determine high-resolution structures of both peptides in complex with SDS and DPC micelles. Additionally, solid state NMR experiments in oriented lipid bilayers were performed to assess structure and orientation in a bilayer environment and to indicate the impact of the peptide on bilayer organization. Both peptides structure as amphipathic α-helices with hydrophobic residues on one side and polar and positively charged residues on the opposite side. RP-1 shows very subtle structural differences when in complex with SDS (anionic) versus DPC (zwitterionic) micelles. This suggests that its specificity for prokaryotic versus eukaryotic membranes does not derive from peptide structural differences in the two systems, but rather from differences in the details of the peptide-lipid interactions. The 2H solid state NMR data are consistent with IL-8α associating peripherally with POPC bilayers, and penetrating deeper into POPC/POPG bilayers. Additional insight is gained from 31P and 15N spectra of IL-8α in these environments.