Solution NMR Structure and Backbone Dynamics of Partially Disordered Arabidopsis thaliana Phloem Protein 16-1, a Putative mRNA Transporter

Abstract

Although RNA-binding proteins in plant phloem are believed to perform long-distance systemic transport of RNA in the phloem conduit, the structure of none of them is known. Arabidopsis thaliana phloem protein 16-1 (AtPP16-1) is such a putative mRNA transporter whose structure and backbone dynamics have been studied at pH 4.1 and 25 °C by high-resolution nuclear magnetic resonance spectroscopy. Results obtained using basic optical spectroscopic tools show that the protein is unstable with little secondary structure near the physiological pH of the phloem sap. Fluorescence-monitored titrations reveal that AtPP16-1 binds not only A. thaliana RNA (K ∼ 67 nM) but also sheared DNA and model dodecamer DNA, though the affinity for DNA is ∼15-fold lower. In the solution structure of the protein, secondary structural elements are formed by residues 3-9 (β1), 56-62 (β2), 133-135 (β3), and 96-110 (α-helix). Most of the rest of the chain segments are disordered. The N-terminally disordered regions (residues 10-55) form a small lobe, which conjoins the rest of the molecule via a deep and large irregular cleft that could have functional implications. The average order parameter extracted by model-free analysis of N relaxation and {H}-N heteronuclear NOE data is 0.66, suggesting less restricted backbone motion. The average conformational entropy of the backbone NH vectors is -0.31 cal mol K. These results also suggest structural disorder in AtPP16-1.

Negative Thermal Expansion and Disorder-to-Order Collapse of an Intrinsically Disordered Protein under Marginally Denaturing Conditions

“…The isotope sources were 15 NH 4 Cl and/or 13 C glucose depending on whether double or single isotope labeling was required. The conditions for cell growth, harvesting and lysis, and further processing toward purification of the protein have also been described . The protein is finally obtained in 50 mM acetic acid, pH 3.5.…”

“…The production of recombinant At PP16-1 has been described earlier . To label the nitrogen and carbon atoms with 15 N and 13 C isotopes, we used the general procedure of growing cells in M9 medium containing the appropriate isotope source.…”

“…The estimate for the eukaryotic proteome containing disordered proteins projects to the 36–63% range . Our tryst with IDP surfaced when we solved the solution NMR structure of the 156 amino acid plant protein At PP16-1 that binds and plausibly transports RNA in the phloem duct . In brief, the protein is low in the content of hydrophobic amino acids but rich in glycine, polar neutral, and charged amino acids.…”

“…12 surfaced when we solved the solution NMR structure of the 156 amino acid plant protein AtPP16-1 that binds and plausibly transports RNA in the phloem duct. 13 In brief, the protein is low in the content of hydrophobic amino acids but rich in glycine, polar neutral, and charged amino acids. Only ∼19% of the residues form the secondary structure consisting of a single αhelix (residues 96−105) and three antiparallel β-strandsβI, βII, and βIII corresponding to residues 3−9, 56−62, and 133− 135, respectively.…”

An engineered construct of cFLIP provides insight into DED1 structure and interactions

“…terminus have 15 N NOEs significantly below the average value, indicating internal motion on the sub-nanosecond timescale (0.44 and 0.38 respectively). By comparison, proteins that are even partially disordered report { 1 H}, 15 N NOE ratios lower than 0.6 (Sashi et al, 2018). The T 1 relaxation values are consistent across the length of the protein, with an average T 1 value of 859 ± 11 ms.…”