10 pags., 8 figs., 2 tabs., Glycine rich polyproline II helix assemblies are an emerging class of natural domains found in several proteins with different functions and diverse origins. The distinct properties of these domains relative to those composed of α-helices and β-sheets could make glycine-rich polyproline II helix assemblies a useful building block for protein design. Whereas the high population of polyproline II conformers in disordered state ensembles could facilitate glycine-rich polyproline II helix folding, the architectonic bases of these structures are not well known. Here, we compare and analyze their structures to uncover common features. These protein domains are found to be highly tolerant of distinct flanking sequences. This speaks to the robustness of this fold and strongly suggests that glycine rich polyproline II assemblies could be grafted with other protein domains to engineer new structures and functions. These domains are also well packed with few or no cavities. Moreover, a significant trend towards antiparallel helix configuration is observed in all these domains and could provide stabilizing interactions among macrodipoles. Finally, extensive networks of Cα-H···Odouble bondC hydrogen bonds are detected in these domains. Despite their diverse evolutionary origins and activities, glycine-rich polyproline II helix assemblies share architectonic features which could help design novel proteins., This study is part of the project PID2022-137806OB-I00, funded by the Spanish Ministry of Science, Innovation and Universities: MICIN/AEI/10.13039/501100011033/FEDER, UE. The funding agency had no role in the study design or the interpretation of the data., Peer reviewed

15 pages, 8 figures, 1 table, Due to the benefits of tomato as an antioxidant and vitamin source, allergy to this vegetable food is a clinically concerning problem. Sola l 7, a class I lipid transfer protein found in tomato seeds, has been identified as an allergen linked to severe anaphylaxis. However, the role of lipid binding in Sola l 7-induced allergy remains unclear. Here, the three-dimensional structure of recombinant Sola l 7 (rSola l 7) has been elucidated using nuclear magnetic resonance spectroscopy (NMR). Its interaction with free fatty acids has been deeply studied; fluorescence emission spectroscopy revealed that different long-chain fatty acids interact with the protein, affecting the only tyrosine residue present in Sola l 7. On the contrary, no changes in the overall secondary structure were observed after the analysis of the circular dichroism spectra in the presence of fatty acids. Unsaturated oleic and linoleic fatty acids presented higher affinity and promoted more significant changes than saturated or short-chain fatty acids. 1H-15N HSQC NMR spectra allowed to determine the regions of the protein that were modified when rSola l 7 interacts with the fatty acids, suggesting epitope modification after the interaction. For corroboration, IgG and IgE binding to rSola l 7 were assessed in the presence of free fatty acids, revealing that both IgE and IgG binding were significantly lower than in their absence, suggesting a potential protective role of unsaturated fatty acids in tomato allergy., This work was supported by grants PID2020-116692RB-I00 (to Mayte Villalba and Eva Batanero) and PID2022-137806OB-I00 (to David Pantoja-Uceda) fundedby MICIN/AEI/10.13039/501100011033 and cofunded bythe European Regional Development Fund. Jorge Parron-Ballesteros acknowledges a predoctoral UCM-Santander grant (CT58/21), Cristobalina Myorga holds a“Nicolas Monardes” research contract by the Andalusian Regional Ministry of Health (RC-0004-2021), and Gustavo A. Titaux-Delgado a “Juan de la Cierva” post-doctoral grant (FJC2021-047976-I) by MICIN/AEI and bythe European Union NextGenerationEU/PRTR. NMRexperiments were performed in the “Manuel Rico” NMRLaboratory of the Spanish National Research Council(CSIC), a node of the Spanish Large-Scale National Facility for Biomolecular NMR (ICTS R-LRB). We are grateful to Dr. Marta Bruix for her advice and expert assistancewith NMR experiments, and to Dr. Douglas V. Laurents for constructive criticism of the manuscript, Peer reviewed

11 pages, 5 figures, 4 tables, Hydrogen bond cooperativity (HBC) plays an important role in stabilizing protein assemblies built by α-helices and β-sheets, the most common secondary structures. However, whether HBC exists in other types of protein secondary structures such as polyproline II (PPII) helices remains unexplored. This is intriguing, since PPII systems as assembling blocks are continuously emerging across multiple fields. Here, using a combination of computational chemistry tools and molecular modeling corroborated by experimental observables, we characterize the distinct H-bonding patterns present in PPII helical bundles and establish that HBC stabilizes intermolecular PPII helices as seen in other protein assemblies such as amyloid fibrils. In addition to cooperative interactions in canonical CO···HN H-bonds, we show that analogous interactions in non-canonical CO···HαCα H-bonds are relevant in Gly-rich PPII bundles, thus compensating for the inability of glycine residues to create hydrophobic cores. Our results provide a mechanistic explanation for the assembly of these bundles., This work has been funded by grants PID2019-109306RB-I00 & PID2022137806OB-I00(D.V.L.) and PID2020-113907RA-I00 (M.M.) from MCIN/AEI/ 10.13039/501100011033. Co-funded by the European Union (ERC, 101042403– BiFOLDOME). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.The authors also gratefully acknowledge the SGAI-CSIC for its assistance and help while using the DRAGO Supercomputer, Peer reviewed

16 pages, 14 figures, 8 pages, Hydrogen bond cooperativity (HBC) is the phenomenon where the collective strengthening of hydrogen bonds, by mutual polarization and non-additive electrostatic effects, in a network exceeds the sum of individual interactions. HBC has been well-studied in α-helices and β-sheets, where it critically stabilizes amyloid structures. During the last 20 years, several natural proteins were characterized which contain multiple aligned and hydrogen-bonded polyproline II (PPII) helices. HBC was recently reported for these PPII helices when arranged along one parallel layer, but its existence in the more abundant antiparallel or mixed parallel/antiparallel PPII assemblies is still unknown. Utilizing computational approaches validated through experimental observables, we report that both canonical CO···HN and non-canonical CO···HαCα hydrogen bonds exhibit mutual reinforcement, revealing a complex hydrogen bonding scheme that manifests HBC in antiparallel PPII helices and in mixed parallel/antiparallel orientations. These findings have fundamental relevance for understanding protein conformational stability and implications for PPII helices as a structural building block for protein design., This work has been funded by grants PID2019-109306RB-I00 and PID2022-137806OB-I00 (D.V.L.) from MCIN/AEI/10.13039/501100011033. Co-funded by the European Union (ERC,101042403–BiFOLDOME,M.M.). The authors also gratefully acknowledge Antonio DíazPozuelo from IQF-CSIC and the SGAI-CSIC for the assistance and help while using the LADONandDRAGO supercomputers,respectively., Peer reviewed

15 pages, 7 figures, The chaperonin GroEL and its mitochondrial and chloroplastic homologs mHsp60 and Cpn60 are large barrel-like oligomeric proteins. Chaperonins facilitate folding by isolating nascent chains in their hollow interior and undergoing ATP-powered conformational transitions. Due to their vital importance, the structures of GroEL and its homologs were extensively studied by x-ray crystallography and CryoEM, revealing rings containing seven subunits. Each subunit has three folded domains and a 24 residue C-terminal extension. Whereas this C-terminal tail has been reported to bind and stimulate the folding of client proteins, it appears to be blurry or invisible, which suggests disorder. The objective of this study is to characterize conformational preferences in the C-terminal tails of GroEL, mHsp60 and representative Cpn60s using circular dichroism and nuclear magnetic resonance spectroscopies and molecular dynamics simulations. The tails of GroEL and mHsp60 consist of two segments. The first is rich in residues typical of intrinsically disordered proteins (PKNDAADLGA and PKEEKDPGMG in GroEL and mHsp60, respectively) and the second segment consists exclusively (GroEL) or almost entirely (mHsp60) of Gly and Met residues. The spectroscopic results reveal that these C-terminal extensions are not wholly disordered but adopt polyproline II helices whose populations are higher in the second Gly/Met-rich segment. These results are corroborated by MD simulations of GroEL7GroES7 complexes with ADP or ATP, or ATP and a client protein. Whereas the C-terminal segments of chloroplastic chaperonins are Gly-poor, they are rich in proline and also adopt polyproline II helix conformations. These results provide insight into the function of chaperonin C-terminal tails., This study is part of projects PID2019 109306RB-I00 and PID2022-137806OB-I00, funded by the Spanish Ministry of Science, Innovation and Universities: MICIN/AEI/10.13039/501100011033/FEDER, UE. NMR
experiments were performed in the “Manuel Rico” NMRlaboratory (LMR) of the Spanish National ResearchCouncil (CSIC), a node of the Spanish Large-Scale National Facility (ICTS R-LRB). This research project was made possible through the access granted by the Gali-cian Supercomputing Center (CESGA) to its supercom-puting infrastructure. The supercomputer FinisTerrae IIIand its permanent data storage system have been fundedby the NextGeneration EU 2021 Plan de Recuperacion,Transformacion y Resiliencia, ICT2021-006904, and also from the Programa Operativo Plurirregional de España 2014-2020 of the European Regional Development Fund (ERDF), ICTS-2019-02-CESGA-3, and from the Programa Estatal de Fomento de la Investigacion Científica y Técnica de Excelencia del Plan Estatal de Investigacion Científica y Técnica y de Innovacion 2013-2016 (1) Sub-programa estatal de infraestructuras científicas y técnicasy equipamiento of ERDF, CESG15-DE-3114, Peer reviewed

Information about funding sources that supported the collection of the data (including research project
reference/acronym): This study is part of project PID2022-137806OB-I00, funded by the Spanish Ministry of
Science, Innovation and Universities: MICIN/AEI/10.13039/501100011033/FEDER, UE, Peer reviewed

[Data avability statement]
Raw NMR data have been deposited in the Biological Magnetic Resonance Data Bank (BMRB) at https://bmrb.io, reference number 53707 and 53708, and the raw CD spectra and MD simulations data have not been deposited in a public repository; all these datasets are available from the corresponding author upon request., Polyproline II (PPII) helices are extended left-handed secondary structures increasingly recognized for their roles in molecular recognition, signaling and within intrinsically disordered regions of proteins. Despite their functional importance, predicting regions with propensity to form PPII helices from sequence alone remains challenging due to subtle sequence determinants and their frequent misclassification as random coil. Here, we present PolyProline Predictor (PPP), a user-friendly web server (https://rmni.iqf.csic.es/software/polypropre/) for empirical, sequence-based prediction of PPII helices. Unlike machine learning approaches, PPP aligns query sequences against a curated database of experimentally validated PPII helices, providing an interpretable, composition-, and position-sensitive similarity map. PPP successfully identified conserved PPII motifs in diverse proteins, and predicted the presence of similar motifs in regions lacking experimental structures but modeled by AlphaFold as extended PPII conformations, such as glycine-rich plant proteins, mycobacterial PE_PGRS virulence factors, and the "disordered" C-terminal tails of GroEL and its homologs, as well as the amyloid-flanking region of the necroptosis effector RIPK3. Molecular dynamics simulations further supported persistent PPII helical bundles in three glycine-rich mycobacterial proteins and more heterogeneous, transient PPII populations in plant proteins and RIPK3. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy validated these predictions for RIPK3, revealing partially populated PPII conformations flanking its amyloid core. Such motifs may regulate its amyloid assembly, offering structural insight into mechanisms of functional amyloid formation. By combining experimental evidence with interpretable prediction, PPP fills a critical gap in bioinformatics tools and enables systematic exploration of regions with propensity to form PPII helices across proteomes, redefining the structural landscape of low-complexity regions., This work has been funded by grants PID2019-109306RB-I00 & PID2022-137806OB-I00 (D.V.L.) from MCIN/AEI/10.13039/501100011033. Co-funded by the European Union (ERC, 101042403—BiFOLDOME, to M.M.). Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them. NMR experiments were performed in the “Manuel Rico” NMR Laboratory (LMR) of the Spanish National Research Council (CSIC), a node of the Spanish Large-Scale National Facility (ICTS R-LRB). This research project was made possible through the access granted by the Galician Supercomputing Center (CESGA) to its supercomputing infrastructure. The supercomputer FinisTerrae III and its permanent data storage system have been funded by the NextGeneration EU 2021 Recovery, Transformation and Resilience Plan, ICT2021-006904, and also from the Pluriregional Operational Programme of Spain 2014–2020 of the European Regional Development Fund (ERDF), ICTS-2019-02-CESGA-3, and from the State Programme for the Promotion of Scientific and Technical Research of Excellence of the State Plan for Scientific and Technical Research and Innovation 2013–2016 State subprogram for scientific and technical infrastructures and equipment of ERDF, CESG15-DE-3114., Peer reviewed