Dedic, E. | Search bioRxiv

bioRxiv2023

Conformational changes in the Niemann-Pick Type C1 protein NCR1 drive sterol translocation

The membrane protein Niemann-Pick Type C1 protein (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking and the mechanism for sterol translocation has remained unclear. Here we provide evidence that a glycocalyx is indeed present inside isolated Saccharomyces cerevisiae vacuoles, and report four cryo-EM structures of NCR1 in two distinct conformations that elucidate how it moves sterol through the glycocalyx. The two conformations, named "tense" and "relaxed", illustrate movement of sterol through a tunnel formed by the luminal domains. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the transmembrane domains form a motor-unit that sequentially adopts a tense and relaxed conformation to drive changes in luminal/extracellular domains. SIGNIFICANCE STATEMENTNiemann-Pick Type C1 (NPC1, named NCR1 in yeast) proteins play a critical role in sterol homeostasis by facilitating the integration of sterols into membranes of acidic organelles like lysosomes and vacuoles. The inner surface of these organelles membranes is shielded by the glycocalyx. Here, we provide evidence that a glycocalyx is present in vacuoles from Saccharomyces cerevisiae and demonstrate that NCR1 transports sterols across it by undergoing conformational changes. Our structures suggest a transport model where sterol transport is linked to proton-driven changes in the transmembrane region. This work sheds light on the mechanism of NPC1 protein function and has broad implications for understanding lysosomal storage disorders and for mechanisms employed by members of the Resistance-Nodulation-Division (RND) superfamily.

biochemistry↗

bioRxiv2022

Integrative solution structure of a PTBP1-viral IRES complex reveals strong compaction and ordering with residual conformational flexibility

RNA-binding proteins (RBPs) are crucial regulators of gene expression and often comprise well-defined domains interspersed by flexible, intrinsically disordered regions. The structure determination of ribonucleoprotein complexes involving such RBPs is not common practice and requires integrative structural modeling approaches due to the fact that they often do not form a single stable globular state. Here, we integrate data from magnetic resonance, mass spectrometry, and small angle scattering to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1 also called hnRNP I) bound to an RNA which is part of the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). PTBP1 binding to this IRES element enhances translation of the viral RNA. The determined structural ensemble reveals that both RNA and protein experience a strong compaction upon complex formation, get ordered but still maintain a substantial conformational flexibility. The C-terminal RNA recognition motif (RRM4) of PTBP1 rigidifies the complex by binding a single-strand RNA linker and, in turn, is essential for IRES-mediated translation. PTBP1 acts as an RNA chaperone for the IRES, by ordering the RNA into a few discrete conformations that expose the RNA stems to the outer surface of the RNP complex for subsequent interactions with the translation machinery. The conformational diversity within this structural ensemble is likely common among RNP complexes and important for their functionality. The presented approach opens the possibility to characterize heterogeneous RNP structures at atomic level.

biochemistry↗