Structural characterization of the interaction of α-synuclein nascent chains with the ribosomal surface and trigger factor

Abstract

The ribosome is increasingly becoming recognized as a key hub for integrating quality control processes associated with protein biosynthesis and cotranslational folding (CTF). The molecular mechanisms by which these processes take place, however, remain largely unknown, in particular in the case of intrinsically disordered proteins (IDPs). To address this question, we studied at a residue-specific level the structure and dynamics of ribosome-nascent chain complexes (RNCs) of α-synuclein (αSyn), an IDP associated with Parkinson's disease (PD). Using solution-state nuclear magnetic resonance (NMR) spectroscopy and coarse-grained molecular dynamics (MD) simulations, we find that, although the nascent chain (NC) has a highly disordered conformation, its N-terminal region shows resonance broadening consistent with interactions involving specific regions of the ribosome surface. We also investigated the effects of the ribosomeassociated molecular chaperone trigger factor (TF) on αSyn structure and dynamics using resonance broadening to define a footprint of the TF-RNC interactions. We have used these data to construct structural models that suggest specific ways by which emerging NCs can interact with the biosynthesis and quality control machinery.

Quantitative determination of ribosome nascent chain stability

“…However, hydrophobic collapse or other partially folded states are accessible to the incomplete nascent chain, and translation is certainly slow enough such that the incomplete nascent chain has sufficient time for the formation of such potential native and nonnative intermediates. Although some experiments have shown cotranslational folding to increase folding efficiency and speed, it is possible that intermediates that form cotranslationally may be off-pathway and result in nonnative, toxic species (7,30,31). To avoid such conformations, cells may have evolved chaperones, such as trigger factor, to "hold and unfold" proteins as they emerge.…”

Force-profile analysis of the cotranslational folding of HemK and filamin domains: Comparison of biochemical and biophysical folding assays

“…In recent years, a number of new experimental methods for analyzing the cotranslational folding of protein domains have been developed. These include real-time FRET analysis [1], and methods in which nascent polypeptide chains of defined lengths are arrested in the ribosome and their folding status analyzed by, e.g., cryo-EM [2,3], protease resistance [1,[4][5][6][7][8][9], NMR [10][11][12][13][14][15], photoinduced electron transfer (PET) [1,16], folding-associated cotranslational sequencing [17], optical tweezer pulling [18][19][20][21], fluorescence measurements [22], and measuring the force that the folding protein exerts on the nascent chain using a translational arrest peptide (AP) as a force sensor [2,3,9,21,[23][24][25]. Further, coarse-grained molecular dynamics simulations of various flavors can provide detailed insights into cotranslational folding reactions [26][27][28][29][30], especially when coupled with experimental studies [3,26,31].…”

Developing NMR methods for macromolecular machines: Measurement of residual dipolar couplings to probe dynamic regions of the ribosome

“…The analysis of these measurements indicated a constraining effect of the ribosome on the alignment of the CTDs of the bL12 protein, a conclusion that was verified by independent measurements using specific paramagnetic alignment of domains. Importantly, this study has laid the foundations for detailed characterization of modified bL12 variants on the ribosome and also for more detailed structural investigations into the co-translational folding of ribosome-nascent chain complexes 3,4 .…”

“…In recent years, X-ray crystallography and cryo-electron microscopy (cryo-EM) have elucidated the details of high-resolution structures of ribosomes, revealing intricate mechanistic information about their function during the translation process 1,2 . In parallel, NMR-based observations of nascent polypeptide chains emerging from the ribosome are providing unique structural and mechanistic insights into co-translational folding processes 3,4 . In order to develop further solution-state NMR spectroscopy as a technique for structural studies of dynamic regions within large complexes, we have explored the measurement of residual dipolar couplings (RDCs) within intact ribosomes, focusing in particular on the mobile bL12 protein from the GTPase-associated region (GAR) of the prokaryotic 70S ribosome.…”