Evidence of the reduced abundance of proline cis conformation in protein poly proline tracts
Proline is found in a cis conformation in proteins more often than other proteinogenic amino acids, where it influences structure and modulates function, being the focus of several high-resolution structural studies. However, until now, technical and methodological limitations have hampered the site-specific investigation of the conformational preferences of prolines present in poly proline (poly-P) homorepeats in their protein context. Here, we apply site-specific isotopic labeling to obtain high-resolution NMR data on the cis/trans equilibrium of prolines within the poly-P repeats of huntingtin exon 1, the causative agent of Huntington's disease. Screening prolines in different positions in long (poly-P11) and short (poly-P3) poly-P tracts, we found that, while the first proline of poly-P tracts adopts similar levels of cis conformation as isolated prolines, a length-dependent reduced abundance of cis conformers is observed for terminal prolines. Interestingly, the cis isomer could not be detected in inner prolines, in line with percentages derived from a large database of proline-centered tripeptides extracted from crystallographic structures. These results suggest a strong cooperative effect within poly-Ps that enhances their stiffness by diminishing the stability of the cis conformation. This rigidity is key to rationalizing the protection toward aggregation that the poly-P tract confers to huntingtin. Furthermore, the study provides new avenues to probe the structural properties of poly-P tracts in protein design as scaffolds or nanoscale rulers. ; This work was supported by the European Research Council under the European Union's H2020 Framework Programme (2014-2020)/ERC Grant Agreement No. [648030], Labex EpiGenMed, an "Investissements d'avenir" program (ANR-10-LABX-12-01) awarded to P.B., and the GPCteR (ANR-17-CE11-0022-01) to N.S. The CBS is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), two national infrastructures supported by the French National Research Agency (ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). Financial support from the TGIR-RMN-THC Fr3050 CNRS, the Spanish MINECO (CTQ2013-40855-R), and Gobierno de Aragón (research group Aminoácidos y Péptidos E19_20R) for conducting the research is gratefully acknowledged. ; Peer reviewed
